Download D. To increase stability of the drug

Pharmaceutics IV/PH 406
Chemical Targeting
Dr. Hamm-Alvarez/PSC 406A
2-1445/[email protected]
What is a prodrug?
 Prodrugs are derivatives of drugs that can be converted enzymatically or
chemically to the biologically active drug, through the action of
hydrolases, transferases, lipases, oxidoreductases, etc.
 Prodrugs can be designed to enhance the delivery characteristics and
therapeutic value of the parent drug
 Since the prodrug approach is based on chemical modification of drugs,
this is considered a chemical approach to drug targeting. As with any
drug targeting strategies, the prodrug strategy requires constant
balancing between toxicity and specificity
Criteria for characterization as a successful prodrug:
1. ABSORPTION. The prodrug must be readily transported to the target
site and uptake must be reasonably rapid.
2. ACTIVATION. Once at the site, the prodrug must be selectively
cleaved to the active drug relative to its conversion at other sites in the
body.
3. RETENTION. The active drug, once selectively generated at the site,
must be retained by the tissue.
Paths to the discovery of prodrugs:
1. Analysis of metabolites of pharmacologically active drugs
a. Codein (prodrug) is converted to morphine (active drug)
b. Acetylsalicylic acid (prodrug) is converted to salicylic acid
2. Design of prodrugs by chemical modification of drugs with some in vivo
activity. This is a RATIONAL approach to improve the usefulness of an
already useful drug
3. Design of an active prodrug starting from a drug that has no in vivo
activity, but promising in vitro activity.
I. What is the role of hydrophilicity and hydrophobicity in chemical
targeting?
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II. Why use chemical targeting?
A. To alter membrane permeability
Ampicillin: The antibiotic ampicillin is very hydrophilic and does not
easily diffuse across the gastrointestinal epithelium following oral
administration. If its free carboxyl group is esterified, its hydrophilicity is
drastically reduced. Several prodrug derivatives of ampicillin have been
generated by esterification, including Bacampicillin and Pivampicillin,
which are readily converted into ampicillin by esterases primarily in the
gastrointestinal mucosa, and then the active species, Ampicillin, is released
into the bloodstream.
H
C
O
H
C
N
S
+NH3
N
C
OR
O
R = H Ampicillin
R = CH(CH3)OCOOC2H5
R = CH2OCOC(CH3)3
Bacampicillin
Pivampicillin
B. To choose dosage form
Chloramphenicol is an antibiotic that can be given orally but its use is
limited in pediatric applications by bitter taste. To interact with taste
receptors, the drug needs to be solubilized in saliva. When solubility is
reduced by complexing the drug with palmitate, the bitter taste is
eliminated. The prodrug is converted in the gut by pancreatic lipase to
chloramphenicol and palmitate; luckily pancreatic lipase is active even on
the insoluble particles.
Chloramphenicol
palmitate ester
Pancreatic lipase
3
Chloramphenicol
+
Palmitate
C. To alter aqueous solubility
Fosphenytoin sodium (Cerebryx) is a phosphate ester prodrug of phenytoin.
It is a replacement for parenteral therapy with the antiepileptic drug,
phenytoin. Fosphenytoin is supplied as a ready-mixed solution in water
buffered to physiological pH. In contrast, phenytoin has to be administered
in 40% propylene glycol and 10% ethanol, adjusted to pH of 12. The high
pH causes adverse effects at the injection site, so the desired properties of
the prodrug include improved aqueous solubility under the necessary dosage
form to reduce side effects. Fosphenytoin is rapidly converted to phenytoin
by phosphatases present in the liver, red blood cells and other tissues, with a
conversion half life of 8-15 min.
H
H
N
O
N
O
N
CH2
O
O P O
O
N
O
CH2O PO
-3
4
H
O
Fosphenytoin
Phenytoin
D. To increase stability of the drug
Levodopa or L-dopa is a prodrug of dopamine that is used for the treatment
of Parkinson’s disease. Dopamine itself is presystemically activated by
sulfate and glucuronide conjugation, requiring continuous infusion. L-dopa
is transported across the blood brain barrier by a neutral amino acid
transporter and then converted to dopamine by L-dopa decarboxylase. LDopa can be absorbed and activated in non-CNS tissues due to the presence
of the neutral amino acid transporter and the enzyme, decarboxylase, in nontarget tissues. This non-specific absorption and activation is responsible for
most side effects in L-Dopa therapy. An improvement of CNS targeting is
achieved if the blood-brain barrier impermeable decarboxylase inhibitor
(carbidopa) is co-administered. This prevents the activation of L-dopa in
peripheral tissues but not in the brain (Sinemet, Merck , Sharp & Dohme)
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E. To eliminate toxicity
Aspirin (acetylsalicylic acid, a prodrug) is less corrosive than salicylic acid.
Dipivefrin is a pivalic ester prodrug of adrenaline that is given in treatment
of glaucoma by local administration. This ester is 100 times more active in
lowering intraocular pressure than adrenaline, due to more efficient
transport through the cornea which is followed by cleavage of the ester
when it passes from the cornea into the aqueous humor. Because lower
doses of the ester are given, the risks of cardiac side effects due to systemic
adrenaline absorption from tear duct overflow are lowered.
OR
RO
CH
R = H Adrenaline
R = COC(CH3)3 Dipivefrin
5
CH2NH2Cl
III. How are prodrugs activated in a tissue-specific manner?
A. Presence of tissue-specific enzymes
Acyclovir is used in treatment of herpes simplex viruses types 1 and 2, and
also for varicella zoster virus (“shingles”). This is an example of a drug that
is converted into the active form only in the virally infected cells due to the
presence of a specific viral enzyme. The basis for its use is that viruses are
less selective in the phosphorylation of possible nucleotide precursors.
Administration of acyclovir to cells expressing viral enzymes results in
phosphorylation of acyclovir by viral thymidine kinase and production of a
phosphorylated product which is trapped in the cell. This product can be
phosphorylated again by cellular enzymes to yield the active triphosphate
compound, which can be incorporated into viral DNA and disrupt the
replication cycle by inhibiting viral DNA polymerase. Administration of
acyclovir to cells not expressing viral enzymes causes little effect, since the
host enzymes do not modify the prodrug. The selectivity here comes from
the activation process, prodrug accessibility to phosphorylating enzyme and
retention.
O
O
N
HN
H 2N
N
N
N
HN
HSV-TK
H 2N
N
N
P O
HO
O
O
GMP
Kinase
O
O
N
HN
N
HN
CELLULAR
enzymes
H2N
P P
N
N
H2N
P
P O
P
N
N
O
O
O
Valacyclovir: valine ester of acyclovir with better oral bioavailability
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Even if a selective viral converting enzyme is absent, generation of
nucleotide analog triphosphates is a general strategy for treatment of
viruses, particularly if there is a differential selectivity for incorporation of
the nucleotide analog triphosphate by viral rather than cellular polymerases.
The tissue specific enzymes in the case of many second and third generation
antivirals are the polymerases themselves.
Problem: Nucleotide is too polar to enter but sometimes the conversion step
of nucleoside to nucleotide is inefficient. Figures from Zemlicka, BBA
1587:286, 2002.
Approach:
Modification of different pronucleotides with lipophilic
phosphoramidates, which can be intracellularly cleaved by esterases—PPA
pronucleotides
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Modification of methylenecyclopropane
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B. Use of unique environment within target tissue
Hypoxia/Reducing Environment or altered pH
Hypoxic cells are enriched in tumors as a result of poor vascularization.
Hypoxic cells are also targets in cardiovascular disease (ischemia),
neurodegenerative diseases, immune diseases and others. These cells have
low oxygen tension, low nutrient levels and often low pH. Targeting of
hypoxic tumor cells has been most studied because these cells are often
resistent to radiation and chemotherapy. The bioreductive approach
involves the selective release of the active species from the prodrug in the
presence of a reducing environment.
Examples of bioreductive enzymes in hypoxic cells: DT-diaphorase,
NADPH reductase, Cytochrome b5 reductase, xanthine dehydrogenase
Jaffar et al., Adv Drug Del Rev 53, 217, 2001
pH can be similarly utilized, by the triggering of pH sensitive cyclization
reactions in specific prodrugs, and by the use of lowered pH to active and/or
retain prodrugs in specific intracellular compartments (lysosomes, for
instance)
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C. Bring the converting enzyme to the target tissue (*Note, these strategies
blend the chemical targeting and biological targeting approaches!)
ADEPT: Some of the best examples of the use of highly toxic drugs in a
targeted manner as prodrugs use the “ADEPT” strategy (Antibody-directed
enzyme prodrug therapy). This approach targets an antibody-enzyme conjugate
to the target site (usually a tumor, which has some characterized antigens). A
prodrug is then added, which can be locally cleaved at the target site, such that
toxicity to the surrounding tissues can be minimized.
Important points for prodrug selection:
1. the prodrug should have a large differential in cytotoxicity between drug and
prodrug
2. the prodrug should be a good substrate for the enzyme under physiological
conditions
3. mammalian homologues should not be able to cleave the prodrug
4. the biologically active agent should have a short half life
Important points for enzyme selection:
1. Activity under physiological conditions
2. Low immunogenicity
3. No endogenous activity in humans
4. High specific activity (because the amount of enzyme that can be delivered
is low)
5. Examples:
 Carboxypeptidase G2—active with nitrogen mustard derivatives,
which can normally not be utilized due to extreme toxicity.
 Carboxypeptidase A—can cleave methotrexate--alanine,
endogenous CPA is a problem
 Alkaline phosphatase—can cleave a wide variety of very potent
prodrugs including precursors to etoposide, mitomycin,
doxorubicin, phenol mustard phosphate; however, large pool of
endogenous alkaline phosphatase is highly problematic, leading to
non-specific cleavage of prodrug
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Other issues:
 Technical difficulties in generation of an antibody-enzyme conjugate that
retains antigen-binding and enzymatic activities. Typically, conjugation
chemistry based on thioether linkages is most commonly utilized. Fusion
proteins combining antibody-enzyme activities, e. g. “Catalytic Antibodies”
may be possible (see below)
 Immunogenicity of bacterial enzymes (used to avoid problems with
endogenous activity) and non-human antibodies, necessity to use
immunosuppressive drugs
Example of catalytic antibody approach: release of etoposide from novel
prodrug at tumor site by injected catalytic antibody (Shabat et al. PNAS
98:7528, 2001)
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Remaining Issues in this study:
 Targeting of catalytic antibody (injection here, not based on use of tumor
antigen), solution would be further modifying to produce a hybrid or
bifunctional antibody
GDEPT: Because of many of the technical difficulties above, another
approach that has been taken is to introduce the activating enzyme into the
target tissue with a gene therapy approach, and then to introduce the
prodrug. This is also called suicide gene therapy.
Major Issues:
1. Efficient, selective and non-toxic delivery of the suicide gene to the
target tissue
2. Factors involved in prodrug choice remain similar to those for ADEPT
3. Because of limitations in efficiency of foreign gene expression, need a
bystander effect (i.e., active drug activity on adjacent cells); for this
reason some investigations have explored the possibility of external
tethering of the expressed enzyme to better facilitate the bystander
effect
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Kirn et al. Trends Molec Med 8:S68, 2002.
GDEPT systems tested in clinical trials:
Herpes simplex virus thymidine kinase-canciclovir combination
Cytosine deaminase-5-fluorocytosine combination
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Virotherapy (Chemical/Biological Targeting)
Viruses have evolved to infect cells, replicate, induce cell death, release
additional particles and to spread. Replication in tumor tissue leads to
amplification of the input dose, while a lack of replication in normal tissues
is associated with efficient clearance. Since we now understand many
factors required for viruses to appropriately replicate, as well as the factors
promoting carcinogenesis, we can envision the design of novel agents such
as replication-selective oncolytic viruses (virotherapy) as targeted
anticancer therapy.
Virotherapy can consist of modified viruses with infectivity in many cells
(adenovirus) or viruses with inherent tumor sensitivity (Newcastle disease
virus).
Engineered viruses: most work has focused on adenovirus. Adenovirus
replication and cell lysis requires expression of early genes including E1A
and B. One example is the insertion of the prostate-specific antigen (PSA)
promoter/enhancer element upstream of the E1A region of the viral genome.
Expression of PSA is high in prostate tumor cells, enabling activation of
E1A selectively in these cells.
Another case is based on the p53 inactivation observed in most tumors. A
compensatory mutation in adenovirus (deletion of a p53 binding protein)
renders this virus selective for replication in cells lacking p53. The
mechanism of this is not completely understood but the efficacy of this virus
has been demonstrated in phase II trials of p53-minus tumors in head and
neck cancer patients.
The other major signaling modification identified in cancer cells includes
mutations in the RB pathway. Selective adenovirus deletions in regions
which interface with this signaling pathway have enabled the selective
expression of these variants in tumors.
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Combination Virotherapy/GDEPT
The Armed Therapeutic Virus™ program (ONYX Pharmaceuticals) consists
of using selectively replicating adenoviruses to deliver anticancer genes,
such as the genes that make enzymes capable of converting inactive prodrug
molecules into toxic chemotherapeutic agents.
Press Release: Onyx Presents Data on Tumor-Specific Intravenous
Gene Delivery at 11th International Conference on Gene Therapy of
Cancer
RICHMOND, CALIF. (December 13, 2002) – Onyx Pharmaceuticals, Inc.
(Nasdaq: ONXX) today announced the presentation of preclinical data
showing the ability of ONYX-411, a second-generation therapeutic virus, to
specifically deliver anticancer gene products to tumors following systemic
(intravenous) administration. The presentation was given today by Yuqiao
Shen, Ph.D., senior scientist of Onyx Pharmaceuticals at the 11th
International Conference on Gene Therapy of Cancer in San Diego,
California.
“We believe these results provide compelling in vivo evidence that ONYX411, an adenovirus engineered to selectively replicate in tumors with
mutations in the retinoblastoma (RB) pathway, can be used as a safe and
efficient vector for the delivery of gene products to the tumor,” said
Leonard Post, Ph.D., Onyx senior vice president, research and development.
“Onyx’ proprietary technology for inserting the therapeutic gene into the
vector is designed to ensure that the gene is expressed only when the virus
replicates. Since the virus is designed to replicate only in tumors, the gene
expression is restricted to the tumors. Using this technology, we believe we
can increase the cancer-killing properties of our viruses while minimizing
the effect on healthy cells, even after intravenous administration.”
In his presentation, Dr. Shen described experiments in which ONYX-443
was administered intravenously to immunodeficient mice carrying human
tumor xenografts. ONYX-443 is an Armed Therapeutic Virus product
constructed by modifying ONYX-411 to express a gene that makes the
enzyme cytosine deaminase (CD). CD converts a non-toxic prodrug (5fluorocytosine, or 5-FC) into a highly toxic chemotherapeutic agent (5fluorouracil, or 5-FU). Dr. Shen reported that strong expression of CD was
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detected only in tumors, not in liver or other normal tissues, following the
systemic administration of ONYX-443. Moreover, during time course
studies (five days post-virus injection until 24 days post-injection in some
experiments), CD expression in tumors increased with time, presumably due
to the continuous replication and spread of the virus. Expression of CD
appeared to correlate with virus replication in vivo.
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