SUITABILITY OF CHELATING AGENTS POSSESSING NITROGEN DONOR GROUPS AS THERAPEUTIC ANTIDOTES FOR CADMIUM

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SUITABILITY OF CHELATING AGENTS POSSESSING
NITROGEN DONOR GROUPS AS THERAPEUTIC ANTIDOTES
FOR CADMIUM

• Toxicology and Biochemistry Research Section,
  NCOH
• M Gulumian

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OCCUPATIONAL AND ENVIRONMENTAL HEALTH

• Certain properties of heavy metal ions such as
  Cd, Pb, Ni, Hg and Bi make them indispensable in
  various industries.

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• Occupational exposure to these metal ions as well
  as their contamination of the environment becomes
  inevitable.

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• These metal ions are highly toxic to humans and
  small quantities are sufficient to cause serious
  health effects.

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Under normal physiological conditions, metal ions
are classified in vivo as

• Essential metal ions those, which in their
  absence, an organisms cannot survive.
• Major metal ions (High concentrations) - Na, K,
  Ca and Mg.
• Minor or trace metal ions (Low concentrations)
  Fe, Cu, Mn, Zn, Co and Mo.
• Beneficial metal ions needed for normal life
  healthy life but in their absence the organism is
  not threatened Cr and Ni.
• Detrimental metal ions have no proven
  beneficial physiological effects and exert toxic
  responses at all concentrations (i.e. Cd, Pb,
  Hg).

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• The essential and beneficial metal ions in vivo
  exist in a well-defined state of homeostasis.
  Detrimental metal ions competitively interact
  with these metal ions and alter the well-defined
  homeostatic state.

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• The altered homeostasis may partially be restored
  by the administration of higher concentrations of
  the beneficial metal ion with which the toxic
  metal ion interacts. The interaction between
  iron and lead is an excellent example where
  mechanisms of iron uptake influence the avidity
  of lead uptake.

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• Living organisms have developed biological
  ligands that are able to reduce the toxicity of
  these metal ions to a considerable extent.
• Examples Metallothionein and glutathione.

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Within any biological compartment, four metal
ion-containing fractions can be observed

• The non-labile protein fraction - storage
  proteins and metal-activated enzymes.
• The labile protein-bound fraction - transport
  proteins.
• The low-molar-mass (l.m.m.) fraction -
  inorganic anions, amino acids and carboxylic
  acids.
• Free unbound aquated fraction - important for
  exchange between various biomolecules.

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Chelation Therapy

• Is probably the most effective means of managing
  heavy metal ion poisoning.

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THERAPEUTIC CHELATING AGENTS

• The term chelate is derived from the Greek word
  chele meaning crabs claw. It is used to
  describe those complexes in which the ligand
  molecule bonds through at least two donor groups
  so that a ring system is formed.
• Ligands which have a potential to form such rings
  are called chelating agents.
  
  
  
  
  
  
  

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• Effective chelation therapy of toxic metal ions
• requires more than a powerful metal-binding
• properties of the chelating agent. In their
• design, there are various sought-after
• properties, which can be conventionally divided
• into two general categories
• 1. Thermodynamic properties.
• 2. Pharmacological properties

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THERMODYNAMIC PROPERTIES

• 1. Thermodynamic stability.
• 2. Selectivity.
• 3. Charge.
• 4. Kinetics.

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PHARMACOLOGICAL PROPERTIES

• Low toxicity.
• Minimal interactions with essential biological
  molecules.
• Low susceptibility to metabolic degradation.
• Rapid elimination once complexed with the toxic
  metal ion.

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• Favourable route of administration (preferably
  oral).
• Therapeutic effect at low dosage.
• Ability to reach the target site.
• These pharmacological factors are
• determined with animal experimentations.

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THERAPEUTIC CHELATING AGENTS

• - Most of the successful therapeutic agents in
  use today were introduced in the 60s.
• - Since, a number of potentially useful
  therapeutic agents have been prepared and
  tested for more effective and superior toxic
  metal ion antagonists.

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Therapeutic chelating agents used

• - 2,3-dimercapro-1-propanol (BAL) As, Pb, Cu,
  Au
• - Meso-2,3-dimercaptosuccinic acid (DMSA) Pb,
  As, Hg, Cu, Au
• Sodium 2,3-dimercaptopropane-1-sulfonate Hg, Cu,
  Pb, As,
• (DMPS)
  Au
• - Ethelenediaminetetraacetic acid Pb, Pu
• - Diethylenetriaminepentaacetic acid (DTPA) Pu
• - D-penicillamine (PEN) Cu, Pb
• - Desferrioxamine (DFO) Fe, Al, Ga

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Chelating Agents for Cadmium

• The development of effective antidotes for
  cadmium intoxication has proven to be difficult.
  This development has been hindered mainly by its
  binding to intracellular metallothionein (24
  hours post exposure).
• Up to date, there is no acceptable chelation
  therapy for cadmium intoxication in humans.

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In animal experiments, two groups of compounds
are tested

• Dithiocarbamates.
• Vicinal dithiols
• None of these chelating agents are, however,
  used therapeutically mainly due to their low
  stability and high toxicity.

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Newly synthesized chelating agents

• With the aid of molecular mechanics, new
  chelating agents were synthesized and were shown
  to be selective for detrimental metal ions such
  as Cd and Pb.
• The selectivity was confirmed by their high
  stability constants and Computer Simulation
  Studies.

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THP-cyclen (N,N,N,N tetrakis
2-hydroxypropyl 1,4,7,10- tetraazacyclododecan
e)

• HO N N OH
• HO N N OH

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TAA-CYCLEN (N,N,N,N-1,4,7,10-tetrakis
acetamidotetraazacyclododecane

• O O
• N N
• NH2 NH2
• NH2 N N NH2
• O O

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Tests Conducted on THP-cyclen and TAA-cyclen

• Selectivity
• Mobilization (in vitro)
• Mobilization (in vivo)
• Toxicity

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SELECTIVITY Computer Simulation Studies

• As some of the aforementioned thermodynamic
  factors are dependent on chemical speciation,
  they can be evaluated theoretically by computer
  simulation studies.

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Computer Simulation Programmes

• COMICS
• ESTA
• NEUPLOT
• TRIPLOT
• HALTAFALL
• ECCLES

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ECCLES (Evaluation of Constituent Concentrations
in Large Equilibrium Solutions)

• Developed by Peter May (1976).
• The Programme is able to solve the relevant mole
  balance equations in blood plasma and displays
  the resulting species distributions in an ordered
  manner to enable changes in the concentrations of
  the major complexes to be readily monitored.

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• A second programme called MIX is used in
  conjunction to ECCLES to generate estimates of
  the equilibrium constants for the many mixed
  ligand (ternary) complexes that might be
  important in the blood plasma for which no data
  is available.

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• Presently, ECCLES contains a set of
• 10,000 formation constants (at 37 oC and 150 mM
  NaCl), ten metal ions and over 100 ligands.
• Computer models such as ECCLES should be
  regarded as the first of the several steps on the
  way to fully understanding the biological effects
  of chelating agents.

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• The efficacy of a chelating agent for mobilizing
  a metal ion from the labile metal-ligand complex
  pool in blood plasma is usually expressed by the
  PMI (Plasma Mobilizing Index) parameter.

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• Total calculated concentration of l.m.m complex
  species in the presence of the chelating agent
• PMI -----------------------------------------
• Total calculated concentration of l.m.m.
  complex species in normal plasma

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Computer Simulation Studies

• PMI curves for different metal ions with
  THP-cyclen

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• Log PMI curves for cadmium ion with different
  chelating agents.

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Mobilization (In Vitro)

• The ability of THP-cyclen, TAA-cyclen, and PEN
  to mobilize cadmium from BSA, Hb and MT was
  investigated in vitro.
• The complexes formed were separated by exclusion
  chromatography.

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Percentage distribution of Cd between Mt and
THP-cyclen
Incubation Time Cd In MT Peak Cd In THP-cyclen Peak
10 min 29 71
3 hrs 26 74
24 hrs 8 92
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Percentage distribution of Cd between Mt and
TAA-cyclen
Incubation Time Cd in MT Peak Cd in TAA-cyclen Peak
10 mins 25 75
3 hrs 19 81
24 hrs 11 89
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TOXICITY

• The toxicity of THP-cyclen, TAA-cyclen and PEN as
  well as their efficacy in reducing the toxicity
  of cadmium were evaluated using the LC50 toxicity
  test on the larvae of the brine shrimp Artemia
  salina (Meyer et al 1982).

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• The test is carried out by placing 48-hours-old
  nauplii into a petri dish containing 4 ml aerated
  artificial brine water solution with yeast. The
  testing compound was then added to the petri
  dish.
• Once the testing compound has been added, the
  petri dish was incubated under illumination.

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• After 24 hours the surviving nauplii in each
  petri dish were counted.
• The LC50 values were then calculated from the
  percentage nauplii survived and the results were
  reported in mmoles/l as well as in mg/ml.

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COMPOUND TESTED LC50 (mg/l) LC50 (mmol/l)
THP-cyclen 9 600 24
TAA-cyclen gt 20 000 gt 50
PEN 2 682 18
Cd 112 1
THP-cyclen Cd - 50
TAA-cyclen Cd - -
PEN Cd - 0.98
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MOBILIZATION (In Vivo)Efficacy of Chelating
Agents as Antidotes for Cadmium Intoxication in
Rats

• Sprague-Dawley rats (190-200g) were adjusted
  before starting the experiments. They were then
  randomly allocated to six groups
• - Group 1 (Control)
• - Group 2 (Cd only)
• - Group 3 (THP-cyclen only)
• - Group 4 (TAA-cyclen only)
• - Group 5 (Cd THP-cyclen)
• - Group 6 (Cd TAA-cyclen)

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Experimental Design.

• 0 hrs saline or Cd administration (sc).
• 24 hrs water or chelating agent administration
  (intubation).
• 96 hrs, animals sacrificed.
• Blood and urine collected at 0, 24, 48, 72 and 96
  hrs.
• Tissue samples collected on sacrifice.

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Animal Survival Rate

EXPERIMENTAL GROUP SURVIVAL RATE
Group 1 80
Group 2 60
Group 3 100
Group 4 100
Group 5 100
Group 6 100
Total (all groups) 90
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Cd Concentration in Rat Blood
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Cd Concentration in Rat Urine
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Cd Concentration in Rat Faeces
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No Change In

• Cu, Zn, aspartate aminotransferase in blood.
• Cu, Zn, Creatinine, Urea, Alkaline Phosphatase in
  urine.
• Cu and Zn in faeces.

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Cd Concentration in Rat Organs
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Histological Evaluation of Internal Organs

• No histological damage was evident in the liver,
  kidney, heart, lungs and brain of all
  experimental animals.
• In testicular tissues, diffuse necrosis of the
  seminiferous tubules and haemorrhage throughout
  of the interstitial tissues were observed in all
  cadmium-exposed groups.

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Conclusions

• The use of nitrogen donors in the design of
  chelating agents is far more superior in
  chelating Cd than the use of sulphur.
• They are selective to the toxic metal ion such as
  Cd.
• They form complexes with the toxic metal ion at
  high rates.
• The synthesized chelating agents are able to
  mobilize Cd from labile as well as from
  non-labile protein binding sites.
• They are non-toxic and safe to use.
• Can be administered orally.
• Can mobilize extracellular Cd at very high rates.
• Can be used to treat acute Cd intoxication.

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• Disadvantages
• Cannot mobilize intracellularly bound Cd.

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Future Work

• Administration of the chelating agents in
  multiple doses.
• Combination therapy the use of chelating agents
  for mobilizing intracellular and extracellular Cd.

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• Simple modification in the outer arms of the
  chelating agents to create a balance between
  polarity and lipophylicity. For example, a
  single carboxylate may create a single negative
  charge to facilitate entry via channels normally
  used for bile acids. Also the use of surfactant
  drug delivery system commonly available to
  pharmaceutical scientists.

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• The results presented are sufficiently
  encouraging to conclude that with careful
  chemical modification and dosing regimes.
  THP-cyclen and TAA-cyclen may produce effective
  clinical cadmium antidotes for humans in the near
  future.

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• E Casimiro
• R Hancock
• F Marsicano