Pharmaceutical

Biotechnology
 Pharmaceutical Biotechnology

 Biotechnology is the use of microorganisms, plants, animals or parts of them

for the production of useful compounds and Pharmaceutical biotechnology
is concerned as the biotechnological manufacturing of pharmaceutical
products.
 An insight into the nature of the traditional processes was achieved in
about 1870 when Pasteur illustrated that chemical conversions in these
processes were performed by living cells, and thus the traditional
processes should be consider biochemical conversions.
Decades following Pasteur's discovery, biotechnological knowledge increased
when the catalytic role of enzymes for most biochemical conversions became
apparent, based on that knowledge tools became available for the control
and optimization of the traditional processes

A further and very important breakthrough took place after the development
of (Molecular Biology). The notion or concept, brought forward by the pioneers
in the molecular biology in around 1950, that DNA encodes proteins and in this
way controls all cellular processes was the impetus for a new period in
biotechnology.
➢ The fast evolving DNA technologies, after the development of the recombinant
DNA technology in 70th, allowed biotechnologists to control gene expression in
the organisms used for biotechnological manufacturing.
➢ These developed technologies opened new ways for the introduction of foreign
DNA into all kinds of organisms.
➢ Thus genetically modified organisms constructed in this way to open up
completely new possibilities for biotechnology.
 Biopharmaceuticals
Complex biological molecules, commonly known as proteins that usually aim at eliminating the
underlying mechanisms for treating diseases.
Essentially used to make (Complex Larger Molecules) with the help of living
cells (like those found in the human body such as bacteria cells, yeast cells,
animals or plant cells).
Unlike the smaller molecules that are given to a patient through tablets, the
large molecules are typically injected into the patient’s body.
Pharmaceutical Biotechnology Products
Antibodies, Proteins and Recombinant DNA
products
 Antibodies- are proteins produced by white blood cells and are used by
immune system to identify bacteria, viruses and other foreign substances
and to fight them off.
 Monoclonal antibodies- are one of the most exciting developments in pharmaceutical
biotechnology at these recent years. (produced as a result of perpetuating the expression of a single
beta lymphocyte. Consequently, all of the antibody molecules secreted by a series of daughter
cells derived from a single dividing parent beta lymphocyte are genetically identical).
Proteins- made of amino acids or large, complex molecules that do
most of the work in the cells and are required for the structure, function,
and regulation of the body’s tissues and organs.

Protein biotechnology- is emerging as one of the key

technologies of the future for understanding the development of
many diseases like cancer or amyloid formation for better
therapeutic intervention.
Recombinant DNA Technologies
Genetic modification of organisms is done by Fusion of any DNA fragment to DNA
molecules able to maintain themselves by autonomous replication. Such molecules
called replicons
 Recombinant DNA

Plasmid or
Vector
Recombinant DNA technology or DNA cloning
technology:

(Application of plasmids in biotechnology)

 Fusing foreign DNA fragment to the isolated plasmid in order to create a recombinant DNA
molecule called replicons.

 Replicons used as carriers for foreign DNA fragments are

termed vectors (include plasmids from bacteria or yeast, or DNA
from bactriovirus, animal virus or Plant virus).
 Foreign DNA- isolated either from microbial, plant or animal
cell
 Restriction enzyme used to cut DNA at a specific site.
 Ligase enzyme used to close circular recombinant DNA.
- Introduction of recombinant DNA into host cell leads to form
(Transformant).
- Vector replicate in the host, thus all daughter cells will inherit
precise copy (a clone) of the recombinant DNA molecule.
 Monoclonal Antibodies
Typically made by fusing myeloma cells with the spleen from a
mouse that has been immunized with the desired antigen.
 Laboratory animals (mammal, e.g. mice) are first exposed to the antigen
that an antibody is to be generated against. Usually this is done by a
series of injections of the antigen in question, over the course of several
weeks. Once spleen cells are isolated from the spleen the B cells are
fused with immortalised myeloma cells. The myeloma cells are selected
beforehand to ensure they are not secreting antibody themselves and that
they lack the (HGPRT) gene, making them sensitive to the incubation in
HAT medium . Fused cells are incubated in HAT medium for roughly 10 to
14 days. Hence, unfused myeloma cells die, because they lack HGPRT.
Removal of the unfused myeloma cells is necessary because they have
the potential to outgrow other cells, especially weakly established
hybridomas. Unfused B cells die as they have a short life span. In this
way, only the B cell-myeloma hybrids survival. These cells produce
antibodies (a property of B cells) and are immortal (a property of myeloma
cells). The next stage is a rapid primary screening process, which
identifies and selects only those hybridomas that produce antibodies of
appropriate specificity. The B cell that produces the desired antibodies can
be cloned to produce many identical daughter clones. Once a hybridoma
colony is established, it will continually grow in culture medium and
produce antibodies.
 Formulation of Biotech Products
Biopharmaceutical Considerations

1. Sterility
Most proteins are administered parenterally and it should be sterile (But are
sensitive to heat and other sterilization treatments) so cannot withstand
[autoclaving, gas sterilization, or sterilization by ionizing radiation].
Protein pharmaceuticals assembled under aseptic conditions, following established
and evolving rules in the pharmaceutical industry for aseptic manufacture.
2.Viral Decontamination
➢ As recombinant DNA products are grown in microorganisms, these organisms
should be tested for viral contaminants and appropriate measures should be
taken if viral contamination occurs.
➢ Excipients with a certain risk factor, such as blood derived human serum
albumin, should be carefully tested before use and their presence in the
formulation process should be minimized.

3.Pyrogen removal
 Pyrogens are compounds that induce fev er.
 Exogenous pyrogens (pyrogens introduced into the body, not generated by
the body itself) can be derived from bacterial, viral or fungal sources.
Bacterial pyrogens are mainly endotoxins shed from gram negative bacteria.
They are lipopolysaccharides.
 Pyrogen removal of recombinant products derived from bacterial sources
should be an integral part of preparation process:
 Excipients used in the protein formulation should be essentially endotoxins-
free.
 Continuous with Pyrogen removal
 Pyrogen removal of recombinant products derived from bacterial sources
should be an integral part of preparation process:
Ion exchange chromatographic procedures (utilizing its negative charge)
can effectively reduce endotoxins levels in solution.
 For solutions, water for injection (compendial standards) is (freshly) distilled
or produced by reverse osmosis.
Ion exchange chromatography
 Excipients Used in Parenteral
Formulations of Biotech Product

 In a protein formulation (active substance), a

number of excipients selected to serve different
purposes.
 The nature of the protein (e.g. lability-rapid
change or destroyed-) and its therapeutic use
(e.g. multiple injection systems) can make
these formulations quite complex in term of
excipients profile and technology (freeze-
drying, aseptic preparation).
 components found in parenteral
formulations of biotech products
1. Active ingredient
2. Solubility enhancers
3. Anti-adsorption and anti-aggregation agents
4. Buffer components
5. Preservatives and anti-oxidants
6. Lyoprotectants/ cake formers
7. Osmotic agents
8. Carrier system

Note: All of the above are not necessarily

present in one particular protein formulation
2. Solubility Enhancers
 Proteins, in particular those that are non-glycosylated, may have a
tendency to aggregate and precipitate.
 Approaches that can be used to enhance solubility include:

1. Selection of the proper pH and ionic strength conditions

2. Addition of amino acids, such as lysine or arginine (used to

solubilize tissue plasminogen activator, t-PA)

3. Addition of surfactants such as sodium dodecylsulfate, to

solubilize non-glycosylate, IL-2 (interleukin-2) can also help to
increase the solubility.
 Notes
 Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein
involved in the breakdown of blood clots.
 As an enzyme, it catalyzes the conversion of plasminogen to
plasmin, the major enzyme responsible for clot breakdown.
 Because it works on the clotting system, tPA is used in clinical
medicine to treat embolic or thrombotic stroke.
 tPA may be manufactured using recombinant biotechnology
techniques. tPA created by this way may be referred to as
recombinant tissue plasminogen activator (rtPA).

 Interleukin 2 (IL-2) is an interleukin, a type of cytokine

signalling molecule in the immune system.
 It is a protein that regulates the activities of white
blood cells (leukocytes, often lymphocytes) that are
responsible for immunity.
 The mechanism of action of these solubility enhancers

depends on
Type of enhancer and protein involved and is not always fully
understood.
Figure 1: Shows the effect of arginine concentration on the
solubility of t-PA (alteplase) at pH 7.2 and 25oC.

A : type I alteplase
100

90

80

C : 50:50 mixture of
Apparent solubility (mg/ml)

70 type I and type I I alteplase

60

50

40

B : type I I alteplase
30

20

10

0
0 0.05 0.1 0.15 0.2 0.25

Arginine-phosphate (M)
➢ In the above examples aggregation is physical in nature, i.e. based
on hydrophobic and/ or electrostatic interactions between
molecules by formation of covalent bridges between molecules
through disulfide bonds, and ester or amide linkages.

➢ In these cases proper conditions should be found to avoid these

chemical reactions (the figure above clearly indicates the dramatic
effect of this basic amino acid on the apparent solubility of t-PA).
3. Anti-adsorption and anti-aggregation
agents

 Anti-adsorption agents (added to reduce adsorption of the active

protein to interfaces).

 Some proteins normally have hydrophobic sites in the core structure.

They tend to expose hydrophobic sites when an interface is present.

❖ These interfaces can be water/air, water/container wall or interfaces

formed between the aqueous phase and utensils used to administer the
drug (e.g. catheter, needle).
 These adsorbed, partially
unfolded protein molecules form
aggregates, leave the surface,
return to the aqueous phase, form
larger aggregates and
precipitate.

 Example:
The proposed mechanism for
aggregation of insulin in aqueous
media through contact with a
hydrophobic surface (or water-air
interface) is presented in Figure 2.
 Hydrophobic surface

Aqueous solution

crystal

monomer Dimer Tetramer Hexamer

Figure 2 Reversible self-association of insulin, its adsorption to the

hydrophobic interface and irreversible aggregation in the adsorbed
protein film
 Native insulin in solution is in an equilibrium state between
monomeric, dimeric, tetrameric and hexameric form.

 The relative abundance of the different aggregation states depends

on the pH, insulin concentration, ionic strength and specific
excipients (Zn2+ and phenol).

 Suggestion: dimeric form of insulin adsorbs to hydrophobic interfaces

and subsequently forms larger aggregates at the interface.

This adsorption explains why anti-adhesion agents can also act as anti-
aggregation agents.
 Ex: Albumin (strong tendency to adsorb to surfaces) and is therefore added
in relatively high concentration (e.g. 1%) as an anti-adhesion agent to protein
formulations.


Mechanism: albumin competes with the therapeutic protein for binding sites and
prevents adhesion of the therapeutically active agent by combination of its binding
tendency and abundant presence.
  Insulin is one of the many proteins that can form fibrillar
precipitates (long rod-shaped structures with diameters in the
0.1 µm range).

This can be
prevented by:
1. Low concentrations of phospholipids and surfactants (as a
fibrillation-inhibitory effect).
2. The selection of the proper pH to prevent this unwanted
phenomenon.

 Apart from albumin, surfactants can also prevent

adhesion to interfaces and precipitation.

Readily adsorb to hydrophobic interfaces with their own

hydrophobic groups and render this interface hydrophilic
by exposing their hydrophilic groups phase.
 4. Buffer components
Buffer selection is an important part of the formulation process, because
of the pH dependence of protein solubility , physical and chemical
stability.

Buffer systems regularly encountered in biotech

formulations are:
1. phosphate
2. citrate
3. acetate
 The isoelectric point (pI)
pH of a solution at which the net primary charge of a protein becomes
zero.

At a solution pH that is above the pI the surface of the protein is

predominantly negatively charged and like-charged molecules will
exhibit repulsive forces.
At a solution pH that is below the pI, the surface of the protein is
predominantly positively charged and repulsion between proteins
occurs.

At the pI the negative and positive charges cancel, repulsive

electrostatic forces are reduced and the attraction forces predominate.
The attraction forces will cause aggregation and precipitation.

The pI of most proteins is in the pH range of 4-6.

 A good example of importance of
20
the isoelectric point (its
negative logarithm [pH] is
[hGH] mg/ml

10
equal to pI) is the solubility
profile of human growth
Circles = recombinant
5 hGH hormone (hGH, pI around
+ve
Triangles = Met-hGH 5) as presented in Figure 1:
Squares = pituitary hGH
char
ge -ve pI: is the pH at a
1 char
particular molecule
ge
carries no net electrical
3 4 5 6 7 charges (overall charge).
pH Thus molecule is affected
Figure 1. A plot of the solubility of by pH of its surrounding
various forms environment and can
of hGH as a function of pH. The become more positively
closed symbols mean that or negatively charged
precipitate was present in the due to the gain or loss,
dialysis tube after equilibration, respectively, of (H+).
whereas open symbols mean that Such molecules have
no solid material was present, and minimum solubility in
 Even short, temporary pH changes can
cause aggregation. Explain why?

 These conditions can occur, for example, during the freeze-

drying process, when one of the buffer components is
crystallizing and the other is not.
 In a phosphate buffer, Na2HPO4 crystallizes faster than NaH2PO4.

drop in pH during the freezing step.

➢ While other buffer components do not crystallize, but form

amorphous systems and then pH changes are minimized.
 5. Preservatives and Anti-oxidants

Antioxidan
 Methionine,
ts
cysteine, tryptophane, tyrosine and histidine are
amino acids that are readily oxidized.
 Proteins rich in these amino acids are susceptible to oxidative
degradation.

The solution
!!!

1. Replacement of oxygen by inert gases in the

vials helps to reduce oxidative stress.
2. Addition of anti-oxidant such as ascorbic acid
or sodium formaldehyde sulfoxylate can be
considered.
 Preservatives
➢ Certain proteins are formulated in the container designed for
multiple injection schemes.
➢ After administering the first dose, contamination with
microorganism may occur and the preservatives are needed to
minimize growth.
➢ Usually, these preservatives are present in concentrations that
are bacteriostatic rather than bactericide in nature.
➢ Antimicrobial agents mentioned in the USP XXIV are the
mercury-containing pheylmercuric nitrate, thimerosal, p-
hydroxybenzoic acids, phenol, benzyl alcohol and
chlorobutanol.
 Shelf Life of Protein Based Pharmaceuticals
Protein can be stored:
(1) as an aqueous solution
(2) in freeze-dried form
(3) in dried form in a compacted state (tablet).

The stability of protein solutions strongly depends on factors such as

pH, ionic strength, temperature, and the presence of stabilizers.
E.g.: Figure 2 shows the pH dependence of α1-antitrypsin and clearly
demonstrates the critical importance of pH on the shelf-life of
proteins.
 Freeze-Drying of Proteins

 Proteins in solution often do not meet the preferred stability

requirements for industrially pharmaceutical products (>2 years),
even when kept permanently under refrigerator conditions (cold
chain).
 The abundant presence of water promotes chemical and
physical degradation processes.
 Importance of Freeze Drying
 Freeze-drying may provide the desired stability by extending shelf
life. During freeze-drying water is removed via sublimation and
not by evaporation.

it works by freezing the material, then reducing the pressure and

adding heat to allow the frozen water in the material to sublimate.

 Three stages can be discerned in the freeze-drying process:

(1) freezing step
(2) primary drying step
(3) secondary drying step.
Table 1. Three stages in the freeze drying process of protein
formulations.
1. Freezing
The temperature of the product is reduced from ambient
temperature to a temperature below the eutectic temperature (Te),
or below the glass transition temperature (Tg) of the system. A Tg is
encountered if amorphous phases are present.
2. Primary drying
Crystallized and water not bound to protein/excipients is removed
by sublimation. The temperature is below the Te or Tg; the
temperature is for example -40oC and reduced pressures are used.
3. Secondary drying
Removal of water interacting with the protein and excipients. The
temperature in the chamber is kept below Tg and rises gradually,
e.g., from -40oC to 20oC.
 The freeze-drying of a protein solution without the
proper excipients causes, as a rule, irreversible
damage to the protein.
 Table 4.3 lists excipients typically encountered in
successfully freeze-drying protein products:
Table 4.3. typical excipients in a
freeze-dried protein formulation

1. Bulking agents: mannitol/ glycine

➢ Reason: elegance/ blowout prevention

❖ Blowout is the loss of material taken away by the water

vapor that leaves the vial. It occurs when little solid
material is present in the vial.
2. Collapse temperature modifier: dextran, albumin/ gelatine
➢ Reason: prevent increase collapse temperature.
3. Lyoprotectant: sugars, albumin
➢ Reason: protection of the physical structure of the protein.
❖ Mechanism of action of lyoprotectants is not fully
understood. Factors that might play a role are:
Mechanisms of action of lyoprotectants

1. Lyoprotectants replace water as stabilizing agent

(water replacement theory),
2. Lyoprotectants increase the Tg of the cake/ frozen
system
3. Lyoprotectants will absorb moisture from the
stoppers
4. Lyoprotectants slow down the secondary drying
process and minimize the chances for overdrying
of the protein. Overdrying might occur when
residual water levels after secondary drying
become too low.
 Delivery of Proteins

 The parenteral Route of Administration

 Parenteral administration is defined as administration via those routes
where a needle is used, including intravenous (IV), intramuscular (IM),
subcutaneous (SC) and intraperitoneal (IP) injections.

 The blood half-life of biotech products can vary over a wide range. For
example, the circulation half-life of t-PA is a few minutes, while
monoclonal antibodies (MAB) have half-lives of a few days
  One reason to develop modified proteins through site
directed mutagenesis

To enhance circulation half-life.

By expanding the mean residence time for short half-life

proteins (switch from IV to IM or SC administration).

1- changes in disposition which

Have a significant impact on the therapeutic performance of
the drug.
These changes are related to:

i. The prolonged residence time at the IM or SC site of

injection compared to IV administration and enhanced
exposure to degradation reactions (peptidases).

ii. Differences in disposition.

Regarding point 1 (Prolonged residence
time at IM or SC site of injection and the enhanced
exposure to degradation reactions.)

A- For instance, diabetics can become “insulin

resistant” through high tissue dipeptidyl peptidase {DPP-IV} activity .

B- Other factors that can contribute to absorption variation are

related to differences in exercise level of the muscle at the
injection site.

C- The state of the tissue, for instance the occurrence of

pathological conditions, may be important as well.
Regarding point 2 (Differences in
disposition).

Upon administration, the protein may be transported to the

blood circulation

or
through the through the capillary
lymphatics wall at the site of
injection.

Note: The fraction of the administered

dose taking this lymphatic route is
molecular weight dependent.
 Routes of uptake of SC or IM
injected drugs

Blood Capillary wall

Low Mwt drugs

Site of injection

lymph
High Mwt drugs
 Molecular weight of different
proteins

 rIFN alpha-2a (Mw 19 kDa)

 Cytochrome C (Mw 12.3 kDa)
 Inulin (Mw 5.2 kDa)
 FUdR (Mw 256.2 Da)

The following Figure shows:

Cumulative recovery in the efferent lymph from the
right popliteal lymph node following SC administration
into the lower part of the right hind leg of sheep
Correlation between the molecular weight and cumulative recovery
70

60

50
lymph recovery [% of dose]

I FN-α-2a
40

30

Cytochrome C
20

I nulin
10

FUdR

0
0 2 4 6 8 10 12 14 16 18 20
molecular weight [kDa]
 Lymphatic transport takes time (hours) and uptake in the
blood circulation is highly dependent on the injection site.

 On its way to the blood, the lymph passes through

draining lymph nodes and contact is possible between
lymph contents and cells of the immune system such as
macrophages, B- and T-lymphocytes residing in the lymph
nodes.
 The Oral Route of Administration

❑ Oral delivery of protein drugs would be preferable because:

1. It is patient friendly
2. No intervention by a healthcare professional is necessary to administer
the drug.
❑ Not Preferable:
Oral bioavailability is usually very low.
 The two main reasons for failure of uptake after oral administration
1. Protein degradation in the gastrointestinal (GI) tract.
2. Poor permeability of the wall of the GI tract in case of a passive
transport process.
 (protein degradation in the GI tract)

i. The human body has developed a very efficient system to break down proteins in our
food to amino acids, or di- or tri-peptides.
ii. These building stones for body proteins are actively absorbed for use wherever
necessary in the body.
iii. In the stomach pepsins (a family of aspartic proteases) are secreted. They are
particularly active between pH 3 and 5 and lose activity at higher pH
values.
iv . Pepsins are endopeptidases capable of cleaving peptide bonds
distant from the ends of the peptide chain. They preferentially (cleave
peptide bonds between two hydrophobic amino acids).
v. Other endopeptidases are active in the GI tract at neutral pH values,
e.g., trypsin, chymotrypsin, and elastase. They have different peptide
bond cleavage characteristics that more or less complement each
other.
vi. Exopeptidases, proteases degrading peptide chains from their ends,
are present as well. Examples are carboxypeptidase A and B.
viii. In the GI lumen the proteins are cut
into fragments that effectively further
break down to amino acids, di-
and tri-peptides by brush border
(microvillus) and cytoplasmic proteases of
the enterocytes (intestinal absorptive
cells).
 (permeability)

i. High molecular weight molecules do not readily penetrate the intact and
mature epithelial barrier if diffusion is the sole driving force for mass
transfer.
ii. Their diffusion coefficient decreases with increasing molecule size.
iii. Protein are no exception to this rule.
iv. Active transport of intact therapeutic recombinant proteins over the GI-
epithelium has not been described yet.
 Conclusion

The above analysis leads to the conclusion that the oral route of
administration for therapeutic protein is unsuitable if high (or at least constant)
bioavailability is required.
O However, for the category of oral vaccines the
above-mentioned hurdles of degradation and
permeation are not necessarily prohibitive.
O Ex: For oral immunization, only a (small) fraction of
the antigen (protein) has to reach its target site to elicit an
immune response.
O The target cells are B-lymphocyte cells that
produce secretory IgA antibodies.
O and antigen presenting accessory cells
located in Peyer’s patches (macroscopically
identifiable follicular structures located in the
wall of the GI tract).
 Peyer’s patches are overlaid with microfold (M) cells
(separate the luminal contents from the lymphocytes).

 These M cells have little lysosomal degradation capacity and

allow for antigen sampling by the underlying lymphocytes.

 Moreover, mucus producing goblet cell density is reduced over

Peyer’s patches.

 This reduces mucus production and facilitates access to the M

cell surface for luminal contents.

 Attempts to improve antigene delivery via the Peyer’s patches

and to enhance the immune response are made by using
microspheres, liposomes or modified live vectors, such as
attenuated bacteria and viruses.
 Alternative Route of Administration
Parenteral administration has disadvantages
(needles, sterility, injection skill) compared to
other possible routes.
Delivery through nose, lungs, rectum, oral
cavity, and skin have been selected as potential
sites of application.
 The potential pros and cons for
different relevant routes
I. Nasal
Advantage:
1. Easily accessible
2. Fast uptake
3. Proven track record with a number of “conventional” drugs
4. Probably lower proteolytic activity than in the GI tract
5. Avoidance of first pass effect
6. Spatial containment of absorption enhancers [osmolarity & pH] is possible
(when drugs exhibits poor membrane permeability, large molecular size, lack
of lipophilicity and enzymatic degradation by amino peptidases).
Nasal
Disadvantage:
1. Reproducibility (in particular under intranasal
pathologies may affect or capacity for nasal
absorption)
2. Safety (e.g., cilliary movement that propelled
proteins into the throat where it is swallowed and
destroyed by the products of the stomach).
3. Low bioavailability for proteins (Because they are
large molecular weight polar drugs thus they
have low membrane permeability).
II. Pulmonary (intratracheal inhalation or
instillation)
Advantage:
1. Relative easy to access (aerosol or syringe).
2. Fast uptake.
3. Proven track record with “conventional” drugs.
4. Substantial fractions of insulin are absorbed.
5. Lower proteolytic activity than in the GI tract.
6. Avoidance of hepatic first pass effect.
7. Spatial containment of absorption enhancer.
Pulmonary
Disadvantage:
1. Reproducibility (in particular under pathological conditions, smoker/non-
smoker).
2. Safety (e.g., inhaled human insulin [powder or liquid] has been shown to
be more immunogenic than comparator insulins given by S.C. routes;
however, adverse effects of antibody formation demonstrated)
3. Presence of macrophages in the lung with affinity for particulates.
III. Rectal
Advantage:
1. Easily accessible
2. Partial avoidance of hepatic first pass
3. Probably lower proteolytic activity than in the upper parts of GI tract
4. Spatial containment of absorption enhancers is possible
5. Proven track record with a number of “conventional” drugs.

Disadvantage:
Low bioavailability for proteins
IV. Buccal
Advantage:
1. Easily accessible
2. Avoidance of hepatic first pass
3. Probably lower proteolytic activity than in the lower parts of the GI tract
4. Spatial containment of absorption enhancer is possible
5. Option to remove formulation if necessary

Disadvantage:
1. Low bioavailability of proteins
2. No proven track record yet.
V. Transdermal
Advantage:
1. Easily accessible
2. Avoidance of hepatic first pass
3. Removal of formulation if necessary is possible
4. Spatial containment of absorption enhancers
5. Proven track record with “conventional” drugs
6. Sustained/controlled release possible

Disadvantage:
Low bioavailability of proteins
 Conclusion

 The nasal, buccal, rectal, and transdermal routes all have been shown to be
of little clinical relevance if systemic action is required, and if simple protein
formulations without an absorption enhancing technology are used.

 In general, bioavailability is too low and varies too much! The pulmonary
route may be the exception to this rule (because in pulmonary the
absorption was strongly protein dependent, with no clear relationship with
it’s molecular weight).
 In human the drug should be inhaled instead of
intratracheally administered.
 The delivery of insulin to Type I (juvenile onset) and
Type II (adult onset) diabetics has been extensively
studied and clinical phase III trials evaluating
efficacy and safety have been performed or are
ongoing.
 The first pulmonary insulin formulation was
approved by FDA in January 2006 (Exubera®).
 It was taken off the market 2008 because of poor
market presentation
 Many pharmaceutical companies doing
research in the field to develop an
inhalational preparation announced the
termination of product development
following the poor acceptance and risk of
lung cancer of the first US FDA approved
inhaled insulin product, Exubera®.

 This formulation produced cough, dyspnoea

(difficulty in breathing), increased sputum,
and epistaxis (nosebleed), and was
contraindicated in patients with chronic
obstructive pulmonary disease (COPD) and
asthma.
Technosphere insulin: a new
inhaled insulin

 MannKind Corporation has

developed a powdered formulation
of insulin with a higher percentage
of absorption from the lungs. This
product, Afrezza® (Technosphere®
insulin), appears to have overcome
some of the barriers that
contributed to the withdrawal of
Exubera® and is currently under
review by the FDA.
 Technosphere insulin is a new inhaled insulin preparation which
mimics normal prandial insulin release. It decreases post-prandial
blood glucose (PPG) levels and has good glycaemic control with
significantly lesser hypoglycaemia.

 Current data show that this formulation has no impact on pulmonary

function.

 Long-term safety studies with regard to pulmonary function and risk

for development of lung carcinoma need to be monitored.

 The FDA is currently reviewing Technosphere insulin for use in both

type 1 and type 2 diabetes.
Technosphere® insulin
 Approaches to enhance bioavailability of
proteins

Classified according to proposed mechanism of action

1. Increase the permeability of the absorption barrier:
▪ Addition of fatty acids/phospholipids, bile salts, enamine derivatives
of phyenylglycine, ester and ether type (non)-ionic detergents,
saponins, salicylate derivatives of fusidic acid or glycyrrhizinic acid,
or methylated β cyclodextrins
▪ Through iontophoresis
▪ By using liposomes.
2. Decrease peptidase activity at the site of absorption and along
the “absorption route”: aportinin, bacitracin, soybean tyrosine
inhibitor, boroleucin, borovaline.
3. Enhance resistance against degradation by modification of the
molecular structure.
4. Prolongation of exposure time (e.g., bio-adhesion technologies).
Examples of Absorption Enhancing Effects
Effect of glycocholate (absorption enhancer) on
nasal bioavailability of some proteins and peptides.
Molecule No. Of AA Bioavailability (%)

Without With
glycocholate glycocholate

Glucagon 29 <1 70-90

Calcitonin 32 <1 15-20

Insulin 51 <1 10-30

Met-hgH 191 <1 7-8

 Major issues now being addressed are
reproducibility, effect of pathological
conditions (e.g., rhinitis) on absorption and
safety aspects of chronic use.
 Absorption enhancing effects were shown
to be species dependent.
 Pronounced differences in effect were
observed between rats, rabbits, and
humans.