probiotic softgel technology innovation - catalent · probiotic softgel technology innovation:...

Probiotic softgel technology innovation

Stable innovative formulations in the consumer’s preferred dosage form Claudia Valla Product Development Manager, Italy Consumer Health 10.05.11

“© 2011 Catalent Pharma Solutions. All rights reserved.”

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What we will talk about

1. Introduction: the technical challenge and the reason why a softgel

2. The background: product development and product characterization

3. The development: what’s unique about our offer platform

4. Scientific background from our partner Probiotical SpA

Introduction

The technical challenge and the reason why a softgel

Updated 10.05.11

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Probiotic softgel technology Innovation: the story

THE CHALLENGE:

TO ACHIEVE

stable softgel with live probiotic cells

By joining expertise of the specialists:

SOFTGEL TECHNOLOGY EXCELLENCE

PROBIOTIC SCIENCE EXPERTISE

SOFTGEL FORMULATION WITH LIVE

PROBIOTIC CELLS

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Probiotic softgel technology Innovation: challenge key points

PROBIOTIC CELLS (AS ACTIVE INGREDIENT)

• Colonization: Ability to colonize the gut*

• Activity: Strain specific effectiveness supported by clinical trials

SOFTGEL (AS DOSAGE FORM)

• Well known and widely accepted by our consumers as the preferred dosage form*

STABILITY (FINAL PRODUCT OBJECTIVE)

• To reach stable product at room temperature (25°C) for 24 months shelf life

• Individual strain balanced stability

*: Independent research commissioned by Catalent in 3 European Countries and the USA, September 2009

*Del Piano M., et a l. J Clin Gastroenterol 2010; 44:S42-S46

FREE FROM COLD CHAIN!

Gut adhesion

Fridge

Attributes: Positive Softgel

%

Hard-Shell

%

Tablet

%

A good size to swallow 70 38 9

Looks convenient to take 66 41 16

I’d feel comfortable taking 57 29 15

Looks appealing 51 11 5

Appears easy to digest 49 17 8

Looks pure 40 5 6

Looks natural 40 7 5

Expressions Planning Source: Catalent European Research

2009

Attributes: Negative Softgel

%

Hard-Shell

%

Tablet

%

Could get stuck in my throat 5 14 71

Looks uncomfortable to swallow 6 18 68

Independent research commissioned by Catalent in 3 European Countries and the USA, September 2009

Why softgels? The consumer preferred dosage form for VMS supplements – attribution

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Probiotic softgel technology Innovation: why softgels with Catalent?

WHY SOFTGELS NEVER BEFORE?

• Probiotic cells sensitive to temperature, oxygen and water activity

• No suitable technology available

More products.

Supporting your own product line with the broadest experience, deepest expertise and unique technologies to take more products to market, faster.

Reliably supplied.

We have delivered customized global manufacturing, packaging, and integrated supply solutions to top innovators for over 75 years.

Better treatment.

With a wide range of options and delivery platforms, we can optimize your product’s healthy benefits and marketability and its fulfillment of label claims throughout shelf life.

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Why probiotics?

•Fast growing global market

•Steady growth trend in the supplement area

•Science evolving fast

•Versatile applications (different health benefits)

•Good consumer awareness

DEFINITION OF PROBIOTICS:

“live microorganisms which, when administered in adequate amounts, confer a health benefit to the host”

Why PROBIOTICS?

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Microflora intestinal balance: a critical point for overall health

MICROBIAL BALANCE IS VERY DELICATE:

•Once altered in favor of potentially pathogenic bacteria, may lead to dysbiosis.

•Harmful bacteria can penetrate in the gut lumen.

•Pathogenic immune response is triggered.

•Mucosal homeostasis turns into chronic inflammation.

•Intestinal discomfort &

various types of diseases throughout the body.

Adapted from: •Sartor RB. Gastroenterology 2004; 126: 1620-1633 •Julia B Ewaschuk, Levinus A Dieleman. World J Gastroenterol 2006 October 7; 12(37): 5941-5950

Pathogenic bacteria

Beneficial bacteria

A number of environmental and genetic factors influence the balance of beneficial vs aggressive microbes

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How can probiotic supplements help?

Probiotic supplementation may help to re-establish the balance of gut flora in favor of beneficial bacteria.

Gut flora unbalance has critical involvement in many healthy issues

THE NEED:

A targeted probiotic formulation supported by well documented strains + carefully selected supportive actives may contribute to

health promoting in various target areas

GOOD BACTERIA BAD BACTERIA

The background: product development and product characterization

Updated 10.05.11

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Probiotic softgel technology innovation: development steps

PROBIOTIC

CELL

(in the formulation) Colonization: Clinical assessment of enhanced colonizing activity* (5:1) compared with traditional strains

SOFTGEL Developed suitable encapsulating process and relevant suspending system to preserve activity/vitality (pilot batch & full scale)-patent pending

STABILITY

(room temperature)

Stability studies:

-pilot batch T18 projected 24 months

-full scale industrial batch: ongoing

Activity: Selected strains with clinically proven efficacy

Vitality: Developed lipid-based coating able to protect probiotic cells (Patent pending).

Manufacturing

*Del Piano M., et a l. J Clin Gastroenterol 2010; 44:S42-S46

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First developed formulation : Intestinal health

INGREDIENTS 109

CFU*/softgel

Bifidobacterium breve BR 03 (DSM 16604) – lipid coated** 1

Lactobacillus plantarum LP 01 (LMG P-21021) – lipid coated** 1

TOT 2

*CFU: projected 24 months shelf life at room temperature (25°C) on the base of pilot batch (T18)

** BETTER PROTECTION: strains in a lipidic microencapsulated

form with a higher gut colonization efficacy (5:1)

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INTELLECTUAL PROPERTY (IP):

•Probiotical: Microencapsulation Coating patent application •Catalent: Patent application with claims protective to the process for forming a softgel containing a stable formulation containing micro-encapsulated probiotic bacteria has been filed

Fully formulated softgel product manufacture

Pilot batch: the 2 strains result equally balanced (coating protection efficient)

Physical stability data available (6 month accelerated)

Product profile written by our clinical writers (intestinal discomfort)

Reference: **Del Piano M., et al. Evaluation of the intestinal colonization by microencapsulated probiotic bacteria in comparison

with the same uncoated strains. J Clin Gastroenterol 2010; 44:S42-S46.

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Lipidic coating of the probiotic cells: What is it? What is it for?

Probiotic bacterial cells “core”

Lipidic coating (food grade saturated vegetable

fats - melting point < 75°C)

ADVANTAGES:

GOOD

STABILITY

(Cell vitality preservation at room

temperature)

COLONIZATION

(Effectiveness/activity in the gut)

desired "insolubility" of

probiotics in the oil matrix (soft

gelatine capsules).

PROTECTION:

In the softgel

fill matrix

In the body

Uniformly coated

bacterial cells

Technology: fluid bed

•strongly increased survival during the transit across the

GI tract. •To effectively

deliver cells to the appropriate delivery site

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Lipidic coating: chemical/physical benefits for coated strain vs uncoated (stability)

•Water: in the sense of Aw (“water activity”), which is the water fraction present in the food matrix that is potentially available for metabolic purposes.

Aw (water activity):

•Aw ideal for bacterial growth is affected by temperature and nutrient levels (strain specific, but usually much faster at room temperature)

•No/less water available: probiotic cells are less prone to begin and close (=die) their natural life-cycle (preserving vitality at room temperature)

H2O (Aw)

Hydrophilic toxic compounds (potential “probiotic killers”)

O2, Air

PRESERVATION FROM THE CONTACT WITH:

The lipid coating functions like an apolar, water repellent wall

protecting the probiotics from water ingress

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Intestinal colonization efficacy of coated probiotic bacteria: the clinical study

Premises:

•Probiotics are able to exert many different beneficial effects on the human host.

•These effects are mediated by the number of viable cells which reach the gut.

The study:

•A comparison between the intestinal colonization by microencapsulated bacteria and the same uncoated strains has been conducted by a double blind, randomized, cross-over study.

•The study involved 44 healthy volunteers.

Conclusion:

•The microencapsulation technique used in the study is a valid strategy to significantly improve gastroresistance of strains.

The exclusive microencapsulation system Catalent is using is 5 times more effective in reaching the gut in a viable form

(than without microencapsulation) Del Piano M., Carmagnola S., Andorno S., Pagliarulo M., Tari R., Mogna L., Strozzi GP., Sforza F., Capurso L. Evaluation of the intestinal colonization by microencapsulated probiotic bacteria in comparison with the same uncoated strains. J Clin Gastroenterol 2010; 44:S42-S46.

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Full Scale Manufacturing Capability

Full scale industrial batch by Catalent:

• Using full scale equipment train

— Nitrogen protection throughout

• Physical and Active checkpoints evaluation (ongoing):

— Physical parameters qualifying the process

— Activity analysis of the probiotics (CFU/dose, coating efficacy evaluation, individual strain analysis)

Catalent fill material processing protects the coating from damage,

delivering the coated material to the gut effectively! Patent application pending

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Probiotic stability data 18 month real time data on trial batch & 24 months projection

Stability Pointers:

•24 month projections calculated using a simple 1st order kinetics – the expected stability trajectory for Probiotics over the last 6 months of shelf-life.

•In all packaging types a projected potency of 5 billion CFU/g => 2.7 billion CFU/capsule.

•Next stability check-point scheduled after 24 months of storage at 25°C (expected in August 2011) to confirm it

Stability data at 25°C on trial batch(1 gram of capsule fill material is equivalent to 1.82 capsules)

0

5

10

15

20

25

0 5 10 15 20

Time (months)

To

tal c

ells

(1

0^

9 C

FU

/g)

Bulk Bottle BlisterSerie4 Serie5 Serie6

25

Pro

duct

Probiotics with Catalent……….. Innovative protected formulation achieving good stability in the consumer preferred dosage form !

Consumer Need

•A stable convenient probiotic solution in the consumer preferred dosage form*.

Challenge •Probiotics are very sensitive to water Activity (Aw) and temperature.

Unique Technology (IP) •Coated probiotic strains processed with a carefully controlled manufacturing process and high rate of gut colonization. •Catalent has exclusivity access to the coating system and has filed a patent application protecting to the encapsulation process

Scientific Proof of Concept •Samples available •Scale up testing-in progress •Stability data both physical and chemical •Product concept support literature

Development Pipeline VMS and OTC

USP (Unique Selling Propositions)

•Advanced delivered formulation •Safe and well supported product •High rate of gut-colonization (5:1) •Consumer Preferred dosage form* •No Need for refrigeration •Delivery platform with scope for a range of probiotic formulations

Delivering

Advanced formulation

with full support package

* Independent research commissioned by Catalent in 3 European Countries (Italy, France, UK) and the USA, September 2009

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How can we support? Formulation tools that will fast track your unique product development

2. Proactive formulation program:

• Pregnancy combination

• Immune Health

• Intestinal Health second formula

• Omega-3

In program: trial samples, stability package, formulation rationale (considering EFSA claims directive).

1. Advanced development platform for intestinal discomfort formulation

3. Custom-made formulations

Bring to the market a unique product maximizing your chances of success.

• Tools supported by in-house pre-formulation:

— Wide selection of pre-evaluated actives

— Formulation compatibility tested for omega-3 and other suspending oils

— Colored shell ingredients

— Analytical stability program support: strains + actives

— Regulatory product support

• Trial Batch /scale up capability using a patent pending encapsulating process


Scientific background from our partner Probiotical

Dr Luca Mogna – R&D Probiotical Updated 10.05.11


Selection of probiotic strains with specific immunomodulation properties

•There is now substantial evidence that probiotics can also provide benefits by modulating immune functions and regulating the balance between pro-inflammatory and anti-inflammatory cytokines.

•The in vitro evaluation of the immunomodulating effect of two selected probiotic strains, Lactobacillus plantarum LP01 and Bifidobacterium breve BR03, was performed using human Peripheral Blood Mononuclear Cells (PBMCs) according to the following scheme:

1. Study of the optimal growth conditions of the probiotic cells (LP01 and BR03 strains);

2. Study of the optimal conditions for the co-culture of probiotic strains with PBMCs (including their quantitative ratio);

3. Study of the immunomodulating effects of LP01 and BR03 strains on PBMCs (induction of cell proliferation, modulation of the phenotypic expression, synthesis of cytokines and immunoglobulins).

Nicola S., et al. Interaction between probiotics and human immune cells. Agrofood 2010; 21:9-12

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0

50

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150

200

IL-1

2p

70

(p

g/m

l)IL

-10

(p

g/m

l)

IL-4

(p

g/m

l)IF

N-

(pg/m

l)

p< 0.05

p< 0.0001

LPS PHA

LPS PHA

p< 0.0001p< 0.05

p< 0.0001 baseline

L. plantarum LP 01

LMG P-21021

Lactobacillus plantarum LP01 •Mean ± S.E.M. of 10

independent experiments.

•Statistical significance is

calculated using the paired

Student’s t-test. Calculated

with respect to baseline

(unstimulated PBMC), p values

< 0.05 are considered

statistically significant.

•The production of IL-12p70

and IL-10 was evaluated in the

culture supernatants after 1

day of stimulation.

•The production of IFN-γ and

IL-4 was evaluated in the


days of stimulation.

The significant induction of an anti-inflammatory response: L. plantarum LP01

Nicola S., et al. Interaction between probiotics and human immune system: two strains with reverse immunomodulatory activities – Poster presented during 5th Probiotics, Prebiotics & New Foods Conference, Rome, Sept 2009.

Bifidobacterium breve BR03

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IL-1

2p

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(p

g/m

l)IL

-10

(p

g/m

l)

IL-4

(p

g/m

l)IF

N-

(pg

/ml)

baselinep< 0.001

p< 0.01

p< 0.01

LPS PHA

PHA PHA

p< 0.001p< 0.001

p< 0.01

B.breve BR 03

(DSM 16604)

The significant induction of an anti-inflammatory response: B. breve BR03

Mean ± S.E.M. of 10

independent experiments.

Statistical significance is

calculated using the paired

Student’s t-test. Calculated

with respect to baseline

(unstimulated PBMC), p values

< 0.05 are considered

statistically significant. The

production of IL-12p70 and IL-

10 was evaluated in the


day of stimulation.

The production of IFN-γ and

IL-4 was evaluated in the


days of stimulation.


Th1 Th2

Th2

0,00

0,10

0,20

0,30

0,40

0,50

pro

/reg

rati

o

baseline B.breve

BR 03

(DSM 16604)

pro

/reg

rati

o

p< 0.001

0,0

0,1

0,2

0,3

0,4

0,5

0,6

baseline L. plantarum

LP 01

LMG P-21021

p<0.001

Lactobacillus plantarum LP01 and Bifidobacterium breve BR03: an unmatched anti-inflammatory probiotic combination



The first human efficacy trial (2004)

•24 subjects in the group receiving mixed L. plantarum LP01 and B. breve BR03 (5 x 109 CFU/strain/day), 20 subjects in the placebo group

•The main parameters evaluated were abdominal pain at different locations (RLQ, LLQ, epigastrium, back and other sites) and other symptoms typically associated with Irritable Bowel Syndrome (constipation, diarrhea, bloating, flatulence, nausea, cephalea and dyspepsia)

Check-points after 14 and 28 days of intake of probiotic formulation or placebo

Almost the totality of parameters recorded a statistically significant improvement in the group receiving LP01 and

BR03 compared with the placebo

Saggioro A. Probiotics in the treatment of irritable bowel syndrome. J Clin Gastroenterol. 2004 Jul;38(6 Suppl):S104-6

The second human efficacy trial (2010)

•110 subjects in the group receiving mixed L. plantarum LP01 and B. breve BR03 (2.5 x 109 CFU/strain/day), 80 subjects in the placebo group

•The main parameters taken into account were aimed at assessing the intestinal motility function (number of weekly evacuations; feces consistency; ease of expulsion; sensation of complete emptying; anal itching, burning, or pain during or after defecation) and abdominal bloating, often associated with Irritable Bowel Syndrome

Check-points after 15 and 30 days of intake of probiotic formulation or placebo

Almost the totality of parameters recorded a statistically significant improvement in the group receiving LP01 and

BR03 compared with the placebo

Del Piano M. et al. The use of probiotics in healthy volunteers with evacuation disorders and hard stools: a double-blind, randomized, placebo-controlled study. J Clin Gastroenterol. 2010 Sep;44 Suppl 1:S30-4

Further in vitro evidence: the inhibition of E. coli by L. plantarum LP01

Reference: Probiotical’s patent number WO 2010/136891 A1: “Bacteriocin producing lactobacillus pentosus and the use thereof in food and pharmaceutical compositions”.

L. plantarum LP01 has a strong inhibition activity on the two E. coli strains tested (ATCC 25922 and ATCC 8739).

LR06 + LP01

LR06

LP02

LP01

Mix of 5 L. plantarum

LP01 + LP02

LP01 + LP02 + LR06

LF5

1) E. coli strain ATCC 25922.

MRS pH 4.3

ID 094

LF5

ID 126

LRE01

Mix of 5 L. plantarum

LP02

LP01

2) E. coli strain ATCC 8739. It’s interesting to note that also washed cells and supernatants maintained a good inhibition activity, thus suggesting that at least part of this action is probably mediated by a specific bacteriocin.

Scientific evidence supporting LP01 and BR03 combination: a final overview

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•Probiotic strains are able to exert many beneficial effects on human health. Each effect is strain-specific and indissolubly related to the features of the strain itself.

•L. plantarum LP01 and B. breve BR03 are able to significantly modulate the immune system, thus inducing an anti-inflammatory environment driven by Interleukin-10 (IL-10).

•The in vitro evidence has been confirmed by two different human efficacy studies aimed at assessing the efficacy of this strain combination in the alleviation of IBS symptoms and restoration of a physiological intestinal motility function.

•Moreover, L. plantarum LP01 demonstrated a good antagonistic activity on 4 different E. coli strains. This could be the basis of its use in a food supplement able to antagonize some potential intestinal pathogens, thus restoring and maintaining a balanced microbiota.



Saggioro A. Probiotics in the treatment of irritable bowel syndrome. J Clin Gastroenterol. 2004 Jul;38(6 Suppl):S104-6

Del Piano M. et al. The use of probiotics in healthy volunteers with evacuation disorders and hard stools: a double-blind, randomized, placebo-controlled study. J Clin Gastroenterol. 2010 Sep;44 Suppl 1:S30-4

Reference: Probiotical’s patent number WO 2010/136891 A1: “Bacteriocin producing lactobacillus pentosus and the use thereof in food and pharmaceutical compositions”.

Possible new areas of interest (1)

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•Oxidative stress – three probiotic strains have been selected for their capability to synthesize glutathione and superoxide dismutase (SOD), thus exerting a significant antioxidant activity. An animal model study has been performed on Wistar rats and highlighted a relevant increase in the blood total antioxidant activity (TAA) and reduced glutathione (GSH).

•Hypercholesterolemia – six probiotic strains have been selected for their capability to synthesize Bile Salt Hydrolases (BSH) or reduce the absorption of cholesterol in the gut by means of complementary ways. An animal model study has been performed on Wistar rats and highlighted a significant reduction of blood LDL cholesterol and a relevant improvement of total cholesterol/HDL cholesterol ratio. An efficacy human trial will be started in June 2011.

Data on file – publication in progress


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•Acute Respiratory Infections (ARI) – five probiotic strains have

been selected for their capability to significantly reduce the incidence,

severity and duration of ARI during three different human efficacy trials

conducted by Prof. Fabrizio Pregliasco. Pregliasco F., et al. A New Chance of Preventing Winter Diseases by the Administration of Symbiotic Formulations. Journal of Clinical Gastroenterology, 2008; 42(2): 224-233 •Folates synthesis – three probiotic strains have been selected for their capability to synthesize folates, confirmed by in vitro studies, an animal model trial and a pre-clinical human assessment. The delivery of bioavailable folates to the epithelial cells of the bowel is also very relevant because they act as a key trophic factor.

•Antagonism of different Candida species – three probiotic strains were characterized for their capability to significantly antagonize harmful or pathogenic yeasts belonging to different Candida species. They could be fruitfully used in vaginal or oral formulations as well.

Pompei A. et al. Enhanced folate status in Wistar rats fed folate-overproducing Bifidobacteria. Journal of Nutrition, 2007; 137:2742-2746;

Pompei A. et al. Folate production by Bifidobacteria as a potential probiotic property. Applied and Environmental Microbiology, 2007; 73(1): 179-185;

Strozzi G.P. and Mogna L., Quantification of folic acid in human faeces after administration of Bifidobacterium probiotic strains. Journal of Clinical Gastroenterology 2008;42 Suppl 3 Pt 2:179-84.



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•Rebalance of omega-6/omega-3 fatty acids ratio – at least two probiotic strains have been selected for their capability to quantitatively convert linoleic acid (LA) into conjugated linoleic acid (CLA). A potential use could be the reduction of omega-6 fatty acids dietary intake, thus improving the omega-6/omega-3 ratio.

•Gaseous colic in infants – at least two probiotic strains have been characterized for their capability to directly inhibit different Gram-negative bacteria isolated from colicky infants. Two different human efficacy trials are planned between June and September 2011.




Thank you very much for your

attention!


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CATALENT PHARMA SOLUTIONS 14 SCHOOLHOUSE ROAD SOMERSET, NJ 08873 + 1 866 720 3148 www.catalent.com

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probiotic softgel technology innovation - catalent · probiotic softgel technology innovation:...

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