U.S. patent application number 14/249542 was filed with the patent office on 2014-08-07 for process for the preparation of a sulfated derivative of 3,5-diiodo-o-[3-iodophenyl]-l-tyrosine.
This patent application is currently assigned to BRACCO IMAGING S.P.A.. The applicant listed for this patent is BRACCO IMAGING S.P.A.. Invention is credited to Pier Lucio ANELLI, Maria ARGESE, Valeria BOI, Livio CAVALIERI, Laura GALIMBERTI, Sonia GAZZETTO, Luciano LATTUADA, Fulvia VELLA.
Application Number | 20140221477 14/249542 |
Document ID | / |
Family ID | 44555080 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140221477 |
Kind Code |
A1 |
ANELLI; Pier Lucio ; et
al. |
August 7, 2014 |
Process For The Preparation Of A Sulfated Derivative Of
3,5-Diiodo-O-[3-Iodophenyl]-L-Tyrosine
Abstract
The present invention relates to a process for the preparation
of the mono sodium salt of the derivative
3,5-diiodo-O-[3-iodo-4-(sulphooxy)phenyl]-L-tyrosine (T3S) by
starting from the corresponding phenolic compound, in the presence
of chlorosulfonic acid and dimethylacetamide as a solvent. The so
obtained T3S compound may conveniently be isolated in a pure form
as a solid in good yields. The present invention further relates to
the process for T3S preparation, wherein the starting reagent is T2
and further comprising the formulation of such compound in tablets.
Furthermore, the invention discloses non-radioactive immunoassays
based on T3S derivatives.
Inventors: |
ANELLI; Pier Lucio; (Milano,
IT) ; ARGESE; Maria; (Sedriano, IT) ; BOI;
Valeria; (Strambino, IT) ; CAVALIERI; Livio;
(Milano, IT) ; GALIMBERTI; Laura; (Fara Gera
d'Adda, IT) ; GAZZETTO; Sonia; (Cascinette d'Ivrea,
IT) ; LATTUADA; Luciano; (Bussero, IT) ;
VELLA; Fulvia; (Monza, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRACCO IMAGING S.P.A. |
Milano |
|
IT |
|
|
Assignee: |
BRACCO IMAGING S.P.A.
Milano
IT
|
Family ID: |
44555080 |
Appl. No.: |
14/249542 |
Filed: |
April 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14110237 |
Oct 7, 2013 |
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PCT/EP2012/056274 |
Apr 5, 2012 |
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14249542 |
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Current U.S.
Class: |
514/518 ;
558/37 |
Current CPC
Class: |
C07C 303/24 20130101;
C07D 495/04 20130101; A61K 9/2054 20130101; C07F 1/04 20130101;
A61K 31/198 20130101; C07C 227/16 20130101; C07C 303/24 20130101;
C07C 305/24 20130101; C07C 227/16 20130101; C07C 229/36
20130101 |
Class at
Publication: |
514/518 ;
558/37 |
International
Class: |
C07C 303/24 20060101
C07C303/24 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 29, 2011 |
IT |
MI2011A000713 |
Claims
1. A process for the preparation of a sulfated form of a thyroid
hormone having formula II (T.sub.3S) according to the following
reaction: ##STR00013## wherein: M is an alkaline metal; comprising
the steps of: a) sulfation of a compound of formula I with
chlorosulfonic acid (CSA) in the presence of dimethylacetamide
(DMAC); and b) salification to give a compound of formula II, in an
aqueous solution of an alkaline metal inorganic salt.
2. The process according to claim 1, wherein said inorganic salt is
a sodium salt.
3. The process according to claim 1, wherein the molar ratio
between CSA and the compound of formula I is 4 to 10.
4. The process according to claim 1, wherein in step a) the
concentration of the compound of formula I in DMAC is 0.060 to
0.090 mol/L of DMAC.
5. The process according to claim 1, wherein the sulfation reaction
in step a) is carried out at a temperature below 10.degree. C.
6. The process according to claim 5, wherein the sulfation in step
a) is left to occur for at least 2 hours.
7. The process according to claim 1, wherein the salification
according to step b) is carried out in an aqueous solution of
NaHCO.sub.3.
8. The process according to claim 1, further comprising a step c)
of purification of the compound of formula II by chromatography on
a polymeric adsorbent solid phase, elution with a decreasing
polarity mixture of water and an organic solvent, wherein said step
c) is optionally preceded by a filtration step.
9. The process according to claim 8, wherein said decreasing
polarity mixture is a mixture of water and an organic polar
solvent.
10. The process according to claim 8, wherein after elution, the
solution is concentrated tip to at least 10 g of Formula II
compound/kg, and the solution is brought to pH values of 5.5 to
6.5.
11. The process according to claim 10, wherein the compound of
formula II is obtained as a solid after treatment with an organic
polar solvent.
12. The process according to claim 11, wherein said polar organic
solvent is selected from the group consisting of: acetone, ethanol,
isopropanol and acetonitrile.
13. The process according to claim 11, wherein the compound of
formula II in the solid form is further micronized.
14. The process according to any one of claims 11-13, which
comprises further admixing the solid and/or micronized form of the
compound of formula II, optionally in combination with
levo-thyroxine (T4), with at least one diluent selected from the
group consisting of: cellulose or a derivative thereof, kaolin,
starch and inorganic alkaline salts selected from: calcium and
magnesium carbonate.
15. The process according to claim 14, wherein the mixture of the
compound of formula II and the diluent which is microcrystalline
cellulose, is further admixed with at least one glidant agent
selected from the group consisting of: glycerol dibehenate,
tribasic calcium phosphate, talc, starch and derivatives thereof,
at least one disintegrant selected from the group consisting of:
croscarmellose or derivatives thereof, crospovidone,
polymethylacrylates, maltodextrin, sodium glycolate starch,
pre-gelatinized starch, sodium alginate, and optionally, a
lubricating agent selected from the group consisting of: magnesium
stearate, zinc stearate, colloidal hydrated silica and colloidal
silicon dioxide, and then formulated in tablets by direct
compression of the mixture.
16. The process according to claim 1, wherein the reagent of
formula I is obtained by iodinating a 3',5 di-iodothyronine (T2)
with an iodinating agent in an aqueous media and in the presence of
an aliphatic amine.
17-27. (canceled)
28. The process according to claim 1, wherein M is Na.
29. The process according to claim 3, wherein the molar ratio is 7
to 9.
30. The process according to claim 5, wherein the sulfation
reaction in step a) is carried out at a temperature comprised from
-10.degree. C. to 8.degree. C.
31. The process according to claim 9, wherein in said mixture of
water and an organic polar solvent the ratio of water to polar
solvent is 1.0:0 to 0.7:0.3.
32. The process according to claim 16, wherein the iodinating agent
is a mixture comprising I.sub.2/NaI.
Description
FIELD OF THE INVENTION
[0001] The field of the present invention relates to a process for
the preparation of sulfated derivatives of thyroid hormones or
salts thereof.
BACKGROUND OF THE INVENTION
[0002] Thyroid hormone tri-iodothyronine
(3,5-diiodo-O-[3-iodophenyl]-L-tyrosine or T3) is the metabolically
most active thyroid hormone. Like thyroxine (T4) it is
physiologically produced by thyroid and stored together with it,
under the form of a thyroglobulin, a glycoprotein precursor. On
average, one thyroglobulin molecule contains three or four T4
residues and, at the most, one T3 residue. TSH production activates
thyroglobulin proteolysis through the enzymes cathepsin D, B and L
with the release of thyroid hormones T3 and T4. However, T3
generation is not limited to this mechanism: actually, in the
peripheral tissues, thyroxine is transformed into tri-iodothyronine
(80% of tri-iodothyronine is peripherally produced by thyroxine and
20% is produced inside thyroid gland).
[0003] The importance of T3 is not only the one due to the fact of
being the most active thyroid hormone. Actually, in this respect,
various pathological conditions are known that are caused by its
deficiency. In particular, e.g., in nervous tissue during embryonal
development and childhood, T3 deficiency gives rise to a reduction
in cerebral and cerebellar cortex growth, axons proliferation, cell
migration, myelinization, dendrite branching and synapse genesis.
As a result of T3 deficiency in the initial stages of life, a delay
in the nervous system development is observed followed by a
cognitive and motor deficit, that may cause a clinical picture
referred to as cretinism. Also in adults it has been demonstrated
by cerebral PET that, when the tri-iodothyronine levels are
reduced, the blood flow inside the brain and glucose cerebral
metabolism are lower. These data may explain the psychomotor
deficit in the hypothyroid individuals.
[0004] In addition to the effects observed in the nervous tissue,
also the ones in the bone tissue are known where the endochondral
ossification is stimulated by tri-iodothyronine, thus rendering the
bone linearly longer through maturation of the epiphysis bone
centers. Even if not necessary after birth for the bone linear
growth, tri-iodothyronine is essential for the proper fetus bones
development.
[0005] Furthermore, T3 effects in the epidermis tissues have been
substantiated, where tri-iodothyronine not only takes part in its
maturation and of skin adnexa, but also in degradation thereof thus
promoting cell regeneration. Therefore, both the excess and the
deficiency of this hormone can cause dermatological problems.
[0006] Therefore, T3 thyroid hormone may definitely be considered
as a pleiotropic hormone, with well documented effects, in addition
to the ones above mentioned, in the blood tissue, where it
increases erythropoietin production and, consequently,
haemopoiesis; in fat tissues, where it promotes maturation of
pre-adipocytes to adipocytes, increases the fatty acids lipolysis
and finally also regulating cholesterol metabolism. Hypothyroidism,
very frequently generated by autoimmune pathologies, is rather
common: actually, prevalence in Italian people is about 1.5% among
females and 1% among males. It is pharmacologically treated in a
satisfactory way through substitutive therapies, mainly based on
synthetic levo-thyroxine (T4), drug of choice because of the very
short half-life of the more active form, i.e. T3, which, for this
reason, cannot be routinely used.
[0007] However, also the therapy with levo-thyroxine shows some
disadvantages connected to the fact that while plasmatic
euthyroidism is restored, the tissutal one not always does. The
study of pharmacological alternatives, such as the ones proposable
on the basis of the thyromimetic T3 activity described in EP
1560575 B, might represent a desirable alternative to the present
treatments of choice.
[0008] However, as far as T3S is involved, the major obstacle seems
to be represented by the difficulties met by a large scale
synthesis. Actually, until now it has been possible to produce T3S
only on a laboratory scale. In this respect, the preparation of T3S
from T3 by means of sulphating agents e.g. concentrated sulphuric
acid (H.sub.2SO.sub.4) or chlorosulfonic acid (CSA) in large excess
has been described, for example in U.S. Pat. No. 2,970,165 and
Biochim. Biophys. Acta, 33, 461 (1959), that describe the
preparation of T3S from T3 in solid form, by means of the direct
addition of concentrated sulfuric acid, at low temperatures.
[0009] Endocrinology, Vol. 117, No. 1, 1-7 (1985) and
Endocrinology, Vol. 117, No. 1, 8-12 (1985) envisage the synthesis
of T3S from T3 by means of the addition under cooling of a
chlorosulfonic acid (CSA) solution in dimethylformamide, followed
by a purification step through Sephadex LH-20.
[0010] Up to now however, none of the prior art processes can be
scaled up for grams production of the final product in a pure form,
mainly because the reported purification procedures need extremely
high volumes.
[0011] Advantageously, is has now been found that the sulfation
reaction starting from tri-iodothyronine with chlorosulfonic acid
(CSA) as a sulfating agent, in the presence of DMAC, offers high
conversion rates. Moreover the purification can be carried out with
smaller volumes than the ones reported in the known prior-art
processes. Eventually, the product T3S can be purified up to the
required levels for its clinical use both for the necessary quality
and quantity (hundreds of grams), also under conditions applicable
on an industrial scale.
[0012] Furthermore, since only radioactive assays to detect T3S
levels in serum, such as the RIA described in Chopra et al. (J.
Clin. Endocrinol. Metab., 1992, 75: 189-194), have been described
until now, the need exists for safer immunoassays based, for
example, on non-radioactive reagents. The use of such a reagents
would also allow clinical and/or research structures to carry out
these measures. To this aim, non radioactive immunoassays have been
developed and are part of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Panel a) T3S calibration curve by competitive ELISA;
Panel b) DELFIA calibration curve. T3S was assayed at 33.6, 56,
93.3, 155.5, 259, 432, 720, 1200, 2000 .mu.g/mL.
[0014] FIG. 2. Schematic of DTPA-T3S monoamide synthesis.
SUMMARY OF THE INVENTION
[0015] The present invention relates to a process for the
preparation of a mono-cationic salt of
3,5-diiodo-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosine of formula II
(T3S), by starting from
3,5-diiodo-O-(4-hydroxy-3-iodophenyl)-L-tyrosine of formula I or a
salt thereof, according to the scheme:
##STR00001##
[0016] wherein M is an alkali metal, preferably Na, comprising the
steps of:
[0017] a) sulfation of the compound of formula I or of the salts
thereof with chlorosulfonic acid (CSA) in the presence of
dimethylacetamide (DMAC) as a solvent;
[0018] b) salification of the sulfated derivative obtained in a) to
give the compound of Formula II (T3S) by adding the reaction
mixture obtained in a) to an aqueous solution of an alkali metal
inorganic salt, preferably a mono-cationic sodium, even more
preferably NaHCO.sub.3.
According to a particularly preferred embodiment, the compound of
formula I (T3) is obtained by means of the iodination of a compound
of formula III (T2):
##STR00002##
with an iodinating agent, preferably with NaI and I.sub.2, in the
presence of an aliphatic amine, preferably selected from linear
mono alkyl (C.sub.1-C.sub.4) aliphatic amines, among which,
ethylamine is preferred.
[0019] The addition of the iodinating agent is carried out in the
presence of an aqueous solvent, preferably water, at a temperature
preferably lower than 25.degree. C. Preferably, the iodinating
agent is present at a molar ratio comprised between 0.9 and 1.1
mol/mol of compound III (T2).
[0020] Thus the process for the preparation of T3S comprises the
preparation of T3 by means of the iodination of T2 under the
conditions above described and then its sulfation with
chlorosulfonic acid in dimethylacetamide, as better described in
the detailed description.
[0021] Moreover, according to a further aspect, the invention also
comprises the formulation of the active principle, T3S, into
pharmaceutical compositions, preferably solid, wherein T3S,
preferably under a powder form, is mixed with a diluting agent and
then a flowing agent, a lubricating agent, preferably glicerol
dibeenate, and a disaggregating agent, preferably croscaramellose
or the derivatives thereof, are added to the mixture their sieving
and their further mixing with the diluting mixture comprising the
active principle.
[0022] Thus according to this realization, the process comprises a
step where the diluent, for example microcrystalline cellulose, is
added in one or more fractions, their mixing, then the preparation
of a mixture comprising a flowing agent, preferably glicerol
dibehenate, a lubricating agent, preferably magnesium or zinc
stearate, hydrated colloidal silica, colloidal silicon dioxide and
preferably also a disintegrating agent, preferably croscaramellose
or the derivatives thereof; then their sieving and their further
mixing with the mixture comprising the active principle together
with the diluent. Further excipients, stabilizers and diluents
(such as for example calcium carbonate) may then be added and mixed
for a variable time.
[0023] According to a particularly preferred aspect, the invention
further discloses a tablet prepared by the process above described,
comprising T3S as the active principle in a quantity comprised from
1 to 1000 .mu.g and comprising the following diluents, excipients,
glidants and lubricants: calcium carbonate, glycerol dibehenate,
croscarmellose sodium salt, hydrate colloidal silica, magnesium
stearate, microcrystalline cellulose. Preferred quantities for a
single dosage are given in the table below:
TABLE-US-00001 Amount per Tablet Calcium carbonate 20-40 mg
Glycerol 2-15 mg dibehenate Croscarmellose 1-10 mg sodium salt
Hydrate colloidal 0.1-5 mg silica Magnesium 0.01-2 mg stearate
Microcrystalline At least 30 mg cellulose
[0024] A further embodiment of the invention is represented by non
radioactive immunoassays.
[0025] Preferably the immunoassay is an Enzyme Linked Immuno Assay
(ELISA), more preferably a competitive ELISA, more preferably
carried out in a multi-well plate, using as detectable moiety a
fluorescent group or an enzyme (e.g., horseradish peroxidase,
alkaline phosphatase, etc.) or an avidin-derivative detectable
moiety (i.e. biotin).
[0026] As a further development of the T3S non-radioactive
detection assays, reagents have been developed for the Lanthanide
Fluorescence Immuno-Assay. This assay, the synthesized reagents,
and kits for T3S quantitation based on the new reagents, represent
a further object of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Object of the present invention is a process for the
preparation of a sulfated form of the thyroid hormone T3,
3,5-diiodo-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosine (T3S) having
formula II as a mono-cationic salt, by starting from
3,5-diiodo-O-[4-hydroxy-3-iodophenyl]-L-tyrosine of formula I or
from a salified derivative thereof:
##STR00003##
[0028] wherein M is an alkali metal, preferably Na, which comprises
the steps of:
[0029] a) sulfation of the compound of formula I (T3) with
chlorosulfonic acid (CSA) in the presence of dimethylacetamide
(DMAC) as a solvent,
[0030] b) salification of the sulfated derivative obtained in a) to
give the compound of formula II. Salification is generally obtained
by means of the addition of the reaction mixture obtained in a) to
an aqueous solution of an alkali metal inorganic salt, preferably a
sodium salt, even more preferably Na.sub.2CO.sub.3 or
NaHCO.sub.3.
[0031] For the purpose of the present invention by T3S is meant the
compound of Formula II comprising either the sulfated form of
tri-iodothyronine or the mono-cationic salts thereof (Formula II
compound).
[0032] Step a) is carried out by adding CSA to a suspension of T3
in DMAC under cooling, while keeping the solution under a vigorous
stirring.
[0033] Temperature is kept at values lower than about 10.degree.
C., more preferably comprised between -10.degree. C. and 8.degree.
C., more preferably between -8.degree. C. and 6.degree. C., even
more preferably between -5.degree. C. and 5.degree. C.
[0034] The addition of CSA to the suspension is made slowly,
preferably in a period of time comprised from 30 to 60 min
depending on the amount of the reagents employed and preferably
under an inert atmosphere, for example under a nitrogen or argon
atmosphere.
[0035] According to a preferred embodiment, the molar ratio between
CSA and T3 is greater than 4, preferably comprised from 4.5 to 10,
even more preferably comprised from 7 to 9. Even more preferably
comprised from 7.5 to 8.5 mol of CSA/mol of T3. The concentration
of T3 in DMAC, expressed as mol of T3/L of DMAC, is comprised from
0.06 to 0.15 mol/L, more preferably from 0.12 to 0.14 mol/L. It
follows that, the ratio between CSA and solvent may be comprised
from 0.35 to 1.28 mol of CSA/L of DMAC, preferably from about 0.8
to 1.15 mol/L, even more preferably from about 0.96 to 1.1 mol of
CSA/L of DMAC.
[0036] After adding CSA, the mixture is allowed to react for a
period of time not higher than 4-5 hours, generally without
cooling, allowing the temperature to reach room temperature
(20-25.degree. C.).
[0037] Sulfation is generally completed, under the described
conditions, when more than 85%, preferably more than 90%, even more
preferably more than 95% T3 has been converted to T3S.
[0038] According to a particularly preferred embodiment, step a) of
the process foresees the addition of CSA to a T3 solution in DMAC
at a concentration of 0.12-0.14 mol of T3/L of DMAC, with a
preferred ratio of about 8 moles of CSA per mole of T3, at a
temperature comprised from about -5.degree. C. to about 5.degree.
C., in a period of time of 30-40 min. At the end of the addition,
cooling is generally stopped and the temperature is allowed to rise
to room temperature (comprised from about 15 to 25.degree. C.), for
not more than 4-5 hours, preferably not more than 2-3 hours.
[0039] The sulfation mixture is then added according to
salification step b), to an aqueous solution of an inorganic alkali
salt, preferably mono-cationic, wherein Na is particularly
preferred cation, in such an amount as to neutralize the present
chlorosulfonic acid.
[0040] Salification is preferably carried out with an aqueous
solution of sodium carbonate (Na.sub.2CO.sub.3) or sodium hydrogen
carbonate (NaHCO.sub.3), in amounts function of the amount of
chlorosulfonic acid used in the former step, and at least
sufficient to neutralize the pH of the resulting solution. In
general, when Na.sub.2CO.sub.3 is used, an amount of about 1.5
moles per mole of CSA is sufficient. According to this embodiment,
the Na.sub.2CO.sub.3 solution concentration is about 0.7 mol/L of
solution. Under such conditions a solution pH after quenching
comprised between 6.5 and 7.5 is obtained.
[0041] According to this embodiment, the corresponding
mono-cationic salt of the T3S compound obtained, has formula II,
wherein M is preferably Na.
[0042] The addition of the reaction mixture according to step b) is
carried out in a period of time which is variable, typically
comprised from 1 h and 3 h, while keeping a temperature lower than
30.degree. C.
[0043] The T3S compound of formula II, obtained in solution as a
mono-cationic salt according to the step b) above described, is
purified by chromatography, in accordance to a further step c).
Chromatography is previously and optionally preceded by
precipitation and/or filtration, for example gravimetrical or under
vacuum, of the reaction mixture obtained in b), with the aim of
reducing part of the inorganic salts that are formed as
by-products.
[0044] Chromatography (c) is carried out on an adsorbent resin, of
the polymer type. Preferably, such a resin is constituted by a
macro reticular aromatic polymeric matrix. Examples of preferred
resins are XAD.TM. Amberlites.TM., even more preferably
Amberlite.TM. XAD.TM. 1600.
[0045] As well known, before its use, the resin is activated by
means of procedures known in the art, such as, for example,
washings with water, acetone or the like (for a general reference,
see Rohm and Haas in "Laboratory Column Procedures and Testing of
Amberlite and Duolite Polymeric Adsorbents", section "Preparation
of Resins"). In accordance with the process of the invention the
resin is preferably activated with the solvent selected for the
next elution (i.e. acetone or a water/acetone mixture).
[0046] T3S is preferably eluted from the resin by an elution
mixture of solvents with a decreasing gradient of polarity,
starting from the mixture having higher polarity. According to a
preferred embodiment, said elution mixture is at first water,
followed by successive dilutions with a suitable polar organic
solvent, in suitable reciprocal ratios.
[0047] Preferred elution mixtures are represented by
water/acetonitrile and water/acetone in ratios comprised from 1:0
to 0.7:0.3. Preferably the elution mixture is represented by a
mixture of water and acetone in a ratio comprised from 1:0 to
0.85:0.15 and the elution rate through the column is generally
comprised from 0.9 to 1.1 volumes of column/h.
[0048] The fractions eluted from the column and containing the
final product with a purity level higher than 95%, more preferably
higher than 96%, 98%, 99% (measured by analytical methods well
known in the art, such as for example UV detection and analysed by
HPLC analysis) are collected together and the active principle can
be isolated by evaporating the solvent, i.e. under vacuum by
freeze-drying or by other known methods.
[0049] However, according to a preferred embodiment, the eluted
fractions are concentrated for example by partial evaporation under
vacuum up to a concentration of about 10 g/kg of solution.
[0050] At this concentration, the pH of the solution is adjusted to
values lower than 6.5, preferably comprised from 5.5 to 6.5, by
adding a diluted strong inorganic acid solution, preferably one
acid selected between sulfuric acid and hydrochloric acid, being
hydrochloric acid particularly preferred, and utilized in diluted
form at a concentration comprised from 0.9 to 1.1 N.
[0051] The solution is further concentrated about 10-15 times and
T3S can be isolated as a solid for example by freeze-drying,
spray-drying, or, preferably treated with an organic solvent,
preferably of a polar type to be isolated in solid form and then
optionally further micronized.
[0052] Thus, according to this preferred embodiment, the Formula II
compound is isolated in a solid form by treatment with a solvent
selected from the group consisting of: acetone, acetonitrile and
C.sub.1-C.sub.4 alcohols. However other solvents may be employed,
which are selected among: aromatic alkanes, ethers, chlorinated
solvents, esters, dimethylformamide, nitrometane,
dimethylsulfoxide, 2-methoxyethanol, or mixtures thereof, that
allow to obtain a salt in solid form, and isolable.
[0053] Thus, in detail, after chromatography and concentration of
the T3S containing fractions up to a concentration of about 10 g/kg
of solution, pH adjustment to values lower then 6.5, preferably
comprised from 5.5 to 6.5, and further evaporation up to a
concentration of the Formula II compound comprised from 170 to 500
g/kg of suspension or gel, the concentrated solution is treated
with an organic solvent. Preferably, said solvent is a polar
organic solvent selected among: acetone, lower alcohols, such as
for example, ethanol, propanol, isopropanol, and the like, and
acetonitrile, being acetone particularly preferred.
[0054] The addition of acetone to the concentrated T3S solution
occurs at a temperature comprised from 20 to 25.degree. C.,
preferably leaving the mixture under stirring for 1-3 h at a
temperature comprised from 0 to 25.degree. C., in order to let the
solid form of the mono-cationic T3S salt precipitate completely.
The addition of the solvent to the suspension occurs according to
known proportions: when acetone is used, it's added in an amount
comprised between 1-11 g acetone/g T3S, at a temperature comprised
from 20-25.degree. C.
[0055] The mono-cationic derivative of formula II, or more
preferably the sodium salt thereof, is thus obtained in solid form
after separation of the liquid phase from the solid one, for
example by filtration, with a HPLC purity higher than 95%, more
preferably, higher than 96%, 98% or even >99%. Thus, taken as a
whole, the process according to the invention allows to obtain
isolation of the final product (T3S) in high yields (overall yield:
.gtoreq.60%) and with a high purity level (HPLC >99%).
[0056] Actually, advantageously with prior art processes, already
in the sulfation mixture a) in the presence of DMAC, the amount of
by-products is lower than 10%, generally lower than 7%.
[0057] The high conversion percentage in the sulfation reaction and
the following salification allow then to obtain a product in pure
form by an industrially applicable chromatographic step and with
limited volumes. T3S is efficiently separated from the other
by-products and has high purity (>99%) even when it is prepared
in hundreds of grams thus rendering the use of this
tri-iodothyronine derivative in clinical practice possible.
[0058] In order to prepare formulations for clinical use, T3S, in
solid form and with a purity up to 99%, is preferably further
micronized, for example under nitrogen pressure, to reduce the
particle size.
[0059] Particularly preferred is a particle size smaller than 25
.mu.m (at least 90%, more preferably at least 95% of the particles
with dimensions lower than 25 .mu.m) resulting stable for at least
one month when submitted to accelerated stability trials in a
climatic chamber.
[0060] Therefore, according to a preferred aspect of the invention,
the process comprises micronization of the solid T3S in a pure
form, to give particles of the above defined size and the use
thereof to prepare solid formulations, for oral administration.
[0061] According to this aspect, after micronization, T3S is
formulated together with suitable additional components in powder
mixtures, optionally also under granular or microgranular form,
preferably formulated as tablets or pills obtained through direct
compression of the powder mixture.
[0062] The T3S formulation in solid form or more preferably into
tablets, provides to add, to the micronized active principle (or
principles when preferably in combination with levo-thyroxine),
firstly a part of the amount of the necessary final diluent,
preferably 30, 40, or preferably at least 50% of the diluent, and
mixing them to give mixture a).
[0063] Preferred diluent is cellulose or the derivatives thereof
for example microcrystalline cellulose. Other suitable diluting
agents are caolin, starch or alkali inorganic salts such as
magnesium or calcium carbonate. Particularly preferred is calcium
carbonate, more preferably in association with microcrystalline
cellulose.
[0064] Mixture a) is then mixed with a mixture b) comprising
further components, in general: a glidant agent, a lubricating
agent and a disaggregating agent, their sieving and their
successive mixing with mixture a) comprising the active
principle.
[0065] Among the disintegrating agents, particularly preferred is
croscaramellose or its derivatives. Other usable agents to this aim
are crospovidone, polymethacrylates, maltodestrines, starch sodium
glicolate, pre-gelatinized starch, sodium alginate.
[0066] Among glidant agents, particularly preferred is glicerol
dibehenate. Other usable glidants are: tribasic calcium phosphate,
talc, starch or derivatives thereof.
[0067] Among the lubricating agents particularly preferred are
magnesium or zinc stearate, colloidal hydrated silica, colloidal
silicon dioxide. Further excipients, stabilizers and diluents (such
as for example calcium carbonate) may be successively added and
mixed for a variable time. The final mixture is then measured out
and the tablets are preferably prepared by direct compression.
[0068] T3S is present in the solid dosage units in amounts
comprised from 1 and 1000 .mu.g, more preferably from 2.5 to 500
.mu.g, even more preferably from 5 to 250 .mu.g, as the only active
principle, or in combination with other active principles,
preferably T4 (levo-thyroxine). According to this embodiment T4 is
present in amounts comprised from 1 to 800 .mu.g, or from 5-400
.mu.g, more preferably from 10-200 .mu.g. Accordingly then, in the
preparation process of tablets comprising both T3 and T4 as active
principles, these are mixed with the preferred diluent(s) in
mixture a) and further mixed with the other components, in their
turn pre-mixed, as above described. Therefore according to a
preferred aspect the invention discloses a tablet prepared by the
process above described, comprising T3S as the active principle, in
a quantity comprised from 1 to 1000 .mu.g together with the
following additional components:
[0069] the diluent, selected from cellulose or derivatives thereof,
preferably together with a second diluent, preferably calcium
carbonate, up to 35% of the total diluent (w/w); [0070] the
glidant, selected from glycerol dibehnate (most preferred), talc,
silica derivatives among which magnesium trisilicate, amides,
tribasic calcium phosphate, are usually present in the composition
in a quantity range from 1 to 10%, most preferably 4 to 6% (w/w);
[0071] the disintegrant selected from starch, croscarmellose sodium
and crospovidone. Preferred is croscarmellose sodium salt in a
quantity ranging from 0.5 to 10% even more preferably comprised
from 1-5%, most preferably comprised from 2- to 4% (w/w); [0072]
the lubricant selected from magnesium stearate, hydrate colloidal
silica and talc, more preferably magnesium stearate and colloidal
silica, in a total quantity range comprised from 0.1 to 7% even
more preferably the first one comprised from 0.1 to 2% and the
second comprised from 0.5 to 5% (w/w).
[0073] Particularly preferred as excipients are the following
ingredients: calcium carbonate, glycerol dibehenate, croscarmellose
sodium salt, hydrate colloidal silica, magnesium stearate,
microcrystalline cellulose, according to the following preferred
quantities:
TABLE-US-00002 Amount per Tablet Calcium carbonate 20-40 mg,
preferably 25-35 mg, more preferably 30 mg Glycerol dibehenate 2-15
mg, preferably 4-8 mg, more preferably 5 mg Croscarmellose 1-10 mg,
preferably 2-6 mg, more preferably 3.5 sodium salt mg Hydrate
colloidal silica 0.1-5 mg, preferably 0.5-4, more preferably 2 mg
Magnesium stearate 0.01-2 mg, preferably 0.1-1 mg, more preferably
0.5 mg Microcrystalline At least 30 mg cellulose
[0074] In a more preferred embodiment, the composition comprises
2.5 to 500 .mu.g T.sub.3S or more preferably 5-250 .mu.g
T.sub.3S.
[0075] For combination compositions where also T4 is present, T3S
is preferably present in a quantity of from 2.5-500 .mu.g and
T.sub.4 of from 1 to 800 .mu.g, or, even more preferably: T.sub.3S:
5-250 .mu.g and T.sub.4: 5-400 .mu.g, or T.sub.3S: 10-100 .mu.g and
T.sub.4 10-200 .mu.g.
[0076] It is intended that the above quantities refer to single
dosage units, preferably tablets of about 110 mg, preferably for
daily single dosage administration, even though the skilled artisan
may envisage adjustments due to alternative composition forms,
tablet weight and/or therapeutic treatment protocols.
[0077] The tablets according to the preferred embodiment show
optimal dissolution rates (see table below) and an optimal
stability of the active principle(s) (at least 24 months).
[0078] The following properties measured in conditions according to
ICH Guidelines:
TABLE-US-00003 Dissolution test .gtoreq.75% after 45' Moisture
content <10% Resistance to crushing >20N HPLC Title T3S
90-110% HPLC Title T4 (when present) 90-110%
[0079] In the process according to the invention all the reagents
including T3 (compound of formula I), are commercially
available.
[0080] However, according to a particularly preferred embodiment,
T3 is prepared by iodination of a compound of formula III
(3,5-diiodo-thyronine, Levoditi, or T2):
##STR00004##
with an iodinating agent, preferably NaI and I.sub.2, in the
presence of an aliphatic amine, preferably selected among the
mono-alkyl (C.sub.1-C.sub.4) linear aliphatic amines, among which
the preferred is ethylamine. T2 is preferably prepared as
described.
[0081] The addition of the iodinating agent is carried out in the
presence of an aqueous solvent, preferably water, at a temperature
preferably lower than 25.degree. C.
[0082] Preferably the iodinating agent is present at a molar ratio
comprised from 0.9 to 1.1 mol/mol of compound III (T2).
[0083] After iodination, T3 is isolated, preferably by filtration,
as sodium salt, then converted in acid form by re-suspension in
water and acidification with an acid, preferably acetic acid or
sulfuric acid.
[0084] The acid form is isolated, preferably by filtration, again
re-suspended in water to remove salts and filtered.
[0085] T3, as a wet solid, is suspended in N,N-dimethylacetamide,
the suspension is anhydrified and submitted to sulfation
reaction.
[0086] According to a preferred realization, the molar ratio
between CSA and T3 is greater than 4, preferably comprised from 4.5
to 10, even more preferably comprised from 7 to 9. Even more
preferably comprised from 7.5 to 8.5 mol of CSA/mol of T3. The
concentration of T3 in DMAC, expressed as mol of T3/L of DMAC, is
comprised from 0.10 to 0.15 mol/L, more preferably from 0.12 to
0.14 mol/L. It follows that, the ratio between CSA and solvent may
be comprised from 0.58 to 1.28 mol of CSA/L of DMAC, preferably
from 0.89 to 1.15 mol/L, even more preferably from 0.96 to 1.09 mol
of CSA/L of DMAC.
[0087] After adding CSA, the mixture is allowed to react for a
period of time not higher than 4-5 hours, generally without
cooling, allowing the temperature to rise to room temperature.
[0088] Sulfation is generally completed, under the described
conditions, when more than 85%, preferably more than 90%, even more
preferably more than 95% T3 has been converted to T3S.
[0089] According to a particularly preferred embodiment, step a) of
the process foresees the addition of CSA to a T3 solution in DMAC
at a concentration of 0.12-0.14 mol of T3/L of DMAC, with a
preferred ratio of about 8 moles of CSA per mole of T3, at a
temperature comprised from about -5.degree. C. to about 10.degree.
C., in a period of time of 30-40 min. At the end of the addition,
the cooling is generally stopped and the temperature is allowed to
rise to room temperature (comprised from about 15 to 25.degree.
C.), for not more than 4-5 hours, preferably not more than 2-3
hours.
[0090] The sulfation mixture is then added according to
salification step b), to an aqueous solution of an inorganic alkali
salt, preferably di-cationic, wherein Na is a particularly
preferred cation, in such an amount as to neutralize the present
chlorosulfonic acid.
[0091] Salification is preferably carried out with an aqueous
solution of Na.sub.2CO.sub.3 or NaHCO.sub.3, in amounts function of
the amount of chlorosulfonic acid used, at least sufficient to
neutralize the pH of the resulting solution. In general, when
Na.sub.2CO.sub.3 is used, an amount of salt of at least 1.5 moles
per mole of CSA is sufficient. When, according to a particularly
preferred aspect, the inorganic alkali metal salt is
Na.sub.2CO.sub.3, its final concentration is at least 0.7 mol/L
solution. Under such conditions, after quenching, a pH of the
solution comprised from 6.5 to 7.5 is obtained.
[0092] According to this embodiment, the corresponding
mono-cationic salt of the T3S compound obtained, has formula II,
wherein M is preferably Na.
[0093] The addition of the reaction mixture according to step b) is
carried out in a period of time which is variable, typically
comprised from 1 h and 3 h, while keeping a temperature lower than
30.degree. C.
[0094] The T3S compound of formula II, obtained in solution as a
mono-cationic salt according to steps b) and c) as above
described.
[0095] The process according to the invention describes for the
first time, according to the Applicant's best knowledge, the
preparation of T.sub.3S. from either T3 or T2, at a purity of at
least 95%, more preferably, of at least 96%, 98% or >99%, for
clinical use.
[0096] According to a further embodiment, the invention also
relates to a non-radioactive T.sub.3S immunoassay, either based on
colorimetric, fluorescent or chemiluminescent detection.
[0097] Preferably the immunoassay is an Enzyme Linked Immuno Assay
(ELISA), more preferably is a competitive ELISA where increasing
amounts of T.sub.3S. compete for the binding to a solid phase bound
anti-T.sub.3S antibody, (e.g. the polyclonal disclosed in Chopra et
al., J. Clin. Endocrinol. Metab., 1992, 75: 189-194) with a fixed
amount of T.sub.3S conjugated with a detectable moiety, such as a
fluorescent group or an enzyme (e.g. horseradish peroxidase,
alkaline phosphatase, etc.) or an avidin binding-derivative (i.e.
biotin) optionally linked to a detectable moiety.
##STR00005##
[0098] The T.sub.3S derivatives useful for the non radioactive
assays are generally comprised in the general Formula A:
##STR00006## [0099] wherein R is selected from the group consisting
of: [0100] a) a detectable moiety, selected from the group
consisting of: a fluorescent group or an enzyme selected from the
group consisting of: horseradish peroxidase, alkaline phosphatase,
[0101] c) an avidin-binding derivative optionally linked to a
detectable moiety, [0102] d) a lanthanide chelating agent.
[0103] When R is a lanthanide chelating agent the T.sub.3S
derivative is preferably the compound of Formula IV.
[0104] The assay is preferably carried out in a multi-well plate.
Preferably, the detectable moiety is a fluorescent group or an
enzyme (e.g., horseradish peroxidase, alkaline phosphatase, etc.)
or an avidin-binding-derivative (i.e. biotin). According to the
latter embodiment, detection is preferably carried out with an
avidin-derivative, preferably streptavidin comprising an enzyme
such as Alkaline Phosphatase or Horseradish Peroxidase, preferably
HRP, which converts specific substrates into coloured, fluorescent
or chemiluminescent products. The use of biotin-avidin interaction,
combined with the various detection luminescence as techniques for
signal development, allows signal amplification and increased
sensitivity comparable to a RIA test (see i.e. Chopra et al., J.
Clin. Endocrinol. Metab., 1992, 75: 189-194) but without the need
for radioactivity, a clear advantage over the prior art.
[0105] The ELISA assay, the T.sub.3S-derivatives, such as the
biotin derivative their synthesis and kits for T3S quantitation
comprising such reagents, represent a further objects of the
present invention.
[0106] As an alternative embodiment the non-radioactive T.sub.3S
immunoassay is developed for a fluorescence technique, called
Lanthanide Fluorescence Immuno-Assay, described in Hemmila I et al.
Anal Biochem. 1984 March; 137(2): 335-43, by which a sensitivity
from 1-1000 .mu.g/ml T.sub.3S is obtained. This assay developed for
T3S detection, the synthesized reagents, and kits for T3S
quantitation comprising said reagents, represent a further object
of the invention.
[0107] Thus, accordingly, a DTPA-T.sub.3S monoamide
(3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)a-
mino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyros-
ine) of Formula IV, represents a chelating compound according to a
preferred embodiment:
##STR00007##
[0108] Other molecules can be designed and synthesized by an expert
in the field, through conjugation of T.sub.3S with a variety of
chelating moieties, among those suitable for complexation of
lanthanide ions, e.g., nitrilotriacetic acid (NTA),
ethylenediaminetetraacetic acid (EDTA),
ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA),
ethylenediaminedisuccinic acid (EDDS), propanediaminetetraacetic
acid (PDTA), diethylenetriaminetetraacetic acid (DTTA),
diethylenetriaminepentaacetic acid (DTPA), and similar molecules.
Conjugation between the chelating agent and T.sub.3S can be
obtained by a variety of methods known to the expert in the field,
including a direct amide bond formation, as exemplified in
Experimental Part, or the use of bifunctional chelating agents,
that may even be commercial products, such as
(S)-1-p-isothiocyanatobenzyldiethylenetriaminepentaacetic acid
(DTPA isothiocyanate-Invitrogen cat. I24221), or similar
products.
[0109] Suitable lanthanide metals to be used as chelate labels are
selected in the group consisting of: samarium, terbium, dysprosium
and europium. Particularly preferred is the Europium chelate
3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)am-
ino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosi-
ne (Formula V).
##STR00008##
[0110] A schematic of its synthesis is shown in FIG. 2. The process
can be summarized as follows: DTPA di-anhydride is partially
hydrolysed by adding an approximately equimolar amount of water
dissolved in a suitable organic solvent, then the product, mainly
composed of DTPA mono-anhydride is reacted with T.sub.3S, in the
presence of a suitable organic or inorganic base. After solvent
evaporation, the oily residue is diluted with water. The resulting
precipitate is collected, washed with water and dissolved in a
water/acetone mixture. This crude reaction product is purified on a
column of Amberlite XAD1600, or similar resin, developed with
mixtures or gradients of water/acetone. The product containing
fractions are collected and evaporated to dryness, yielding the
desired DTPA-T.sub.3S monoamide. Lanthanide complexation is
obtained according to known procedures by adding an equimolar
amount of a lanthanide salt to the monoamide water solution and
adjusting the pH at 7 with a suitable base (e.g. NaOH). Optionally,
the lanthanide chelated product can be desalted by adsorption on a
resin column (e.g. Amberlite XAD1600) and elution with
water/solvent mixtures.
[0111] Also in this case, a sensitivity comparable to the known RIA
test (see Chopra et al., ibidem) is obtained while avoiding the use
of radioactive isotopes which represents a clear advantage over the
prior art assay. From the above teachings the skilled man may
envisage alternative formats of the ELISA which are nevertheless
comprised in the present invention. For instance, the target
hormone T.sub.3S can be covalently bound to the plate and the
antibody, optionally linked to a tracer enzyme or used in
combination with an antibody linked to a tracer enzyme, used to
competitively measure T3S level in an unknown sample. According to
a further embodiment, the tracer is the antigen itself (T.sub.3S)
directly bound to a detectable enzyme (e.g., Alkaline Phosphatase
or Horseradish Peroxidase) according to procedures known to the
skilled man, also available in ready-to-use conjugation kit.
[0112] According to a further embodiment, the invention comprises a
kit for T.sub.3S administration and dosage in serum, wherein said
kit comprises a dosage kit for T.sub.3S immunodetection by the
above disclosed non-radioactive assays and an
administration/therapeutic kit with a number of T.sub.3S or
T.sub.3S and T4 composition daily doses (i.e. the weekly,
bi-weekly, monthly or bi-monthly need), preferably in the form of
tablets as described above. The dosage kit for T.sub.3S non
radioactive immunodetection may comprise according to a first
preferred embodiment, polyclonal antibodies, the avidin-binding
T.sub.3S derivative, wherein preferably the conjugate is
T.sub.3S-biotin and the avidin-derivative detectable moiety is i.e.
streptavidin. More preferably, the avidin-derivative is
streptavidin and the detectable moiety comprises an enzyme
chemiluminescent moiety (such as Alkaline Phosphatase or
Horseradish Peroxidase), preferably HRP.
[0113] According to the lanthanide fluorescence immunoassay derived
embodiment, the kit may comprise, together with antibodies,
reagents specifically developed for such a detection, such as a
lanthanide metals chelated complex T.sub.3S derivatives, wherein
the metal is selected in the group consisting of: samarium,
terbium, dysprosium and europium. Particularly preferred is the
Europium chelate
3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)am-
ino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosi-
ne (Formula V compound).
[0114] The kit may also comprise T.sub.3S standards for the
preparation of a calibration curve. The standard may be pre-diluted
and ready for use as a solution at the correct concentration or for
solubilisation in a suitable solvent. The kit may also comprise
other reagents selected from the group consisting of: a diluent, a
dye-molecule, a buffer, a preservative, an anti-T.sub.3S antibody,
an instruction leaflet.
[0115] The invention is now described by the following examples
which are only explanatory and must not be construed as limitative
of the scope of the invention.
EXPERIMENTAL SECTION
Example 1
Preparation of T.sub.3S in DMAC
[0116] All the amounts of the raw materials are expressed with
reference to 100 g of T3.
[0117] 3,5-diiodo-O-(4-hydroxy-3-iodophenyl)-L-tyrosine (100 g;
0.154 mol) was suspended in DMAC (2.0 L) under nitrogen atmosphere
and vigorously stirred in order to avoid solid precipitation. After
cooling to -5.degree. C., CSA (142.2 g; 1.229 mol) was added
dropwise in 40 min while keeping the temperature between
-5/5.degree. C. At the end of the addition, cooling was stopped and
the reaction mixture was left under stirring for about 4 h. The
reaction mixture was added dropwise in 1.5 h, into a stirred
aqueous solution of sodium bicarbonate (335.5 g; 3.994 mol in
water, 4.5 L). At the end of the addition, from the so obtained
solution with time it was observed the precipitation of a
crystalline solid as a mixture of inorganic salts. Such a solid was
filtered off, then the obtained solution was purified on
Amberlite.TM. XAD.TM. 1600 by means of elution with water/acetone
mixtures having decreasing polarity collecting the eluate into
fractions. The fractions containing the product having an
appropriate purity level were evaporated under vacuum up to a
concentration of 10 g/kg. The pH of such suspension was adjusted to
6.2 with HCl 1N. The suspension was further concentrated up to a
ratio of about 1:3 solid and residual water. Such a residue was
treated with acetone under cooling for 2 h, then filtered off and
washed with acetone. The product was dried at 40.degree. C. under
vacuum. 74 g of T.sub.3S were obtained as a white solid. Yield on
the anhydrous base: 62%.
Example 2
Preparation of T.sub.3S from T2 (Levoditi)
[0118] The reaction schematic is presented below:
##STR00009##
[0119] All quantities of raw materials are expressed for 1 kg of
Levoditi.
[0120] Iodine (approx. 0.48 kg, source: SQM), NaI (approx. 0.65 kg,
source: Ajay-SQM) and water were charged in a reactor 18-22.degree.
C. and stirred until complete dissolution. The resulting iodinating
mixture was maintained under stirring at room temperature until
use.
[0121] Levoditi obtained from L-thyrosine according to the process
described in: Chalmers, J. R. et al. A. J. Chem. Soc. 1949,
3424-3433), NaI (approx. 0.32 kg) and water were charged in another
reactor and 70% monoethylamine was added.
[0122] The iodinating mixture was added to the reaction
mixture.
[0123] The suspension obtained was stirred for at least 6 h at
18-22.degree. C., then was cooled to 0.degree. C. in 1 h, stirred
for 3-4 h and filtered. The cake was washed with water.
[0124] The wet solid was suspended in water and acetic acid was
added to the mixture and stirred. The suspension was filtered and
the cake washed with water.
[0125] The wet solid was re-suspended in water stirred, filtered
and washed with water.
[0126] The cake was then suspended in DMAC (approx. 12.15 kg) and
the suspension was anhydrified distilling under vacuum.
[0127] The suspension was cooled to 5-10.degree. C. and, in
nitrogen atmosphere, CSA (approx. 1.54 kg) was slowly added and the
temperature maintained below 15.degree. C.
[0128] The solution was heated to 18-22.degree. C. in 1 h and
maintained for another hour, then was added in a reactor containing
a solution of Na.sub.2CO.sub.3 (approx. 2.27 kg) in water (approx.
29.02 kg), previously prepared, maintaining the temperature under
30.degree. C.
[0129] The solution was purified onto a column of Amberlite XAD
1600 (12.5 L) by elution of water (87.5 L) and water/acetone
mixtures (125 L) with decreasing polarity starting from 95:5 to
70:30. The fractions with high HPLC purity were collected and
distilled under vacuum until the desired composition was achieved
(approx. 0.04 kg T.sub.3S/L suspension). The suspension was cooled
to 40.degree. C. and Ethanol (approx. 5.22 kg) was added, obtaining
a solution.
[0130] The mixture was cooled to 0.degree. C. in 2 h, causing
precipitation, stirred for another hour and then filtered. The cake
was washed with Ethanol/water mixture at room temperature.
[0131] Wet solid was dried at approximately 40.degree. C. under
vacuum. 0.98 kg of pure T3-Sulfate sodium salt (HPLC Area %>99%)
were obtained as a white solid.
[0132] Overall yield from T2 (on the anhydrous base): 68.5%.
Example 3
Preparation of T.sub.3S Tablets
[0133] The active principle, also in combination with different
amounts of levo-thyroxine, was pre-mixed for fifteen minutes with
50% of the content of the microcrystalline cellulose.
[0134] To this pre-mixture the following ingredients were added in
this order: glicerol dibehenate, colloidal hydrated silica, sodium
croscaramellose, magnesium stearate and calcium carbonate
(previously sieved through a 0.6 mm clean light/mesh sieve),
together with the remaining 50% of the microcrystalline cellulose,
mixing for further 20 minutes. The uniformity of distribution of
active principle in the mixture was checked by sampling from six
points of the mixer; the text showed that the active principle (or
the active principles) uniformly distribute within the mixture,
also in the case of formulation with levo-thyroxine.
[0135] The powders mixture was then compressed by means of a rotary
tablet press equipped with a round flat punch and the tablets were
submitted to tests for friability, disaggregation times and the
active principle or principles distribution.
[0136] The results of the texts performed on the mixing and
pressing process confirmed reproducibility of both of them, for
T.sub.3S dosages comprised from 25 to 200 .mu.g. Moreover they
showed that the tablets so obtained had parameters corresponding to
the requirements provided for by the official European
Pharmacopoeia (VI Ed.).
Tablet Composition
TABLE-US-00004 [0137] T.sub.3S Na salt 20.6 .mu.g (corresp. to 20
.mu.g T.sub.3S) Calcium carbonate 30 mg Glycerol dibehenate 5 mg
Croscarmellose 3.5 mg sodium salt Hydrate colloidal silica 2 mg
Magnesium stearate 0.5 mg Microcrystalline Up to 110 mg
cellulose
[0138] The tablets prepared as above described were used in
clinical trials Phase I on thyroidectomised individuals, showing
that they can be used as a thyroid hormone replacement therapy (see
US 2011/0245342).
[0139] In fact, T.sub.3S was shown to be absorbed (crossing the
Gastrointestinal Barrier), was found in serum after oral
administration and was converted to the clinically active T3 in a
dose-related fashion. T3 levels in serum were still detectable 48
hrs after single dose administration.
Example 4
Quantitation of T.sub.3S by Immunoassay with Chemiluminescence
Detection
Synthesis of T35 biotin derivative
[0140] Briefly, T3S biotin derivative was synthesized as follows:
N-hydroxysuccinimidyl
d-biotin-15-amido-4,7,10,13-tetraoxapentadecylate A (50 mg; 0.0849
mmol) was solubilized in DMAC (2 mL), to which DIPEA (14.5 uL;
0.0866 mmol) was added, while maintaining the reaction mixture
under continuous stirring at 0.degree. C. T3S (68.4 mg; 0.0908
mmol, prepared as described in Mol & Visser, Endocrinology
1985, 117:1-7) was then added and after a few minutes the
suspension was left to heat up to room temperature to give a clear
solution. It was allowed to stir for 2 h, then kept overnight at
the same temperature. DMAC was evaporated under reduced pressure
(10 mbar; 40.degree. C.) to give a colourless oil. The crude so
obtained was dissolved in H2O and purified by Semi-preparative
HPLC. The fractions containing the product were collected,
concentrated and finally lyophilized to give T3S-biotin as a white
solid (59.6 mg; 0.0495 mmol). Yield 58%. A polyclonal anti-T3S
antiserum was obtained in rabbits as described in Chopra et al., J.
Clin. Endocrinol. Metab., 1992, 75: 189-194.
[0141] The assay was based on a competitive ELISA in which
increasing amounts of T3S competed for antibody binding with a
fixed amount of T.sub.3S conjugated with biotin, in a white 96 well
plate. The employment of the biotin-avidin interaction, which
allows signal amplification, combined with luminescence as
technique for signal development allowed for a sensibility
comparable to the RIA test (described in Chopra et al., J. Clin.
Endocrinol. Metab., 1992, 75: 189-194).
[0142] Standard solutions of T3S were prepared at the following
concentrations: 1000, 200, 40, 8, 1.6 .mu.g/mL in Diluent Buffer:
PBS, 0.05% Tween, 0.3% BSA
[0143] The tracer solution (T3S-Biotin, 180.6 .mu.M) was prepared
in the above diluent buffer. Antibody solution: T3S rabbit
antiserum was diluted 1:50000 in Diluent Buffer plus 8 mM ANS
(1-anilino-8-naphthalene sulfonate), 1.2 mg/mL Sodium
Salicylate.
[0144] A 96 well white plate was coated over night at 4.degree. C.
with 100 .mu.L/well of 2 .mu.g/mL anti Rabbit IgG diluted in
phosphate buffer pH 7.8. At the same time, Standard solutions of
biotin labelled T3S were combined with the diluted antiserum and
the T3S-biotin solution as reported in Table Table A. The mixed
samples were incubated at room temperature in the dark, overnight.
The day after, the plate was washed four times with Washing Buffer
(0.05% Tween 20 in PBS), then incubated in Blocking Buffer (2% BSA
in Washing Buffer) for 1 h at room temperature.
[0145] Afterwards, the plate was rinsed four times with Washing
Buffer, 100 .mu.L/well of the mixed samples were added in
triplicate and the plate was incubated 3 h at room temperature.
[0146] Then, the plate was rinsed three times with Washing Buffer
and incubated with Streptavidin Poly-HRP (10 ng/mL in RASA, 100
.mu.L/well) for 1 h at room temperature.
[0147] After additional six washes, the plate was incubated with
SuperSignal ELISA Femto Maximum Sensitivity Substrate (100
.mu.L/well) for 5 min in the dark and the emitted light was read as
counts per second (CPS) with a luminescence plate reader.
TABLE-US-00005 TABLE A Calibration Curve Preparation T3S/1
Antiserum T3S-biotin T3S/1 (.mu.L) (.mu.L) (.mu.L) CS 5 (1000
pg/mL) 250 125 50 CS 4 (200 pg/mL) 250 125 50 CS 3 (40 pg/mL) 250
125 50 CS 2 (8 pg/mL) 250 125 50 CS 1 (1.6 pg/mL) 250 125 50 B0 --
125 50 NSB -- -- 50
[0148] The calibration curve was prepared in buffer using five
concentrations of the test item in the range 1.6-1000 .mu.g/mL. The
curve is shown in FIG. 1, panel a).
Example 5
Quantitation of T3S by the Lanthanide Fluorescence Immunoassay
Preparation of Formula V Compound
[[3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)a-
mino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyros-
inate(6-)]europate(3-)]trisodium (Formula V)
##STR00010##
[0149] Synthesis of Eu-DTPA-T35 monoamide
[0150] The reaction scheme of the synthesis of
3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)am-
ino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-t
y r o s i n e (DTP A-T3S monoamide) is shown in FIG. 2.
[0151] A solution of H.sub.2O (0.282 ml; 15.64 mmol) in DMAC (43
mL) was added dropwi se to a suspension of
N,N-bis[2-(2,6-dioxylenol orange-4-morpholinyl)ethyl]glycine A
(4.27 g; 11.94 mmol) in DMAC (85 mL) at room temperature. At the
end of the addition the mixture was heated to 80.degree. C. After
4.5 h the reaction mixture was cooled to 25.degree. C. and a
solution of T3S/1 (3 g; 3.98 mmol) and DIPEA (2.71 mL; 15.92 mmol)
in DMAC (85 mL) was added dropwise over 20 min. DMAC was evaporated
under reduced pressure (10 mbar; 40.degree. C.). The oily residue
was diluted with H.sub.2O (200 mL), obtaining precipitation of a
yellowish solid that was filtered washed with H2O and dried. The
crude so obtained was dissolved in Acetone/H2O 20:80 (v/v), the
solution (pH=2.97) was loaded on an Amberlite.RTM. XAD-1600 resin
column (200 mL; diam. 6 cm) and eluted with a Acetone/H.sub.2O
gradient. The fractions containing the product having similar
composition were collected and evaporated to give the ligand
DTPA-T3S as a solid (1.27 g; 1.15 mmol). Yield 26%.
[0152] Europium chloride hexahydrate (0.17 g, 0.46 mmol) was added
in portions to a solution of the ligand DTPA-T3S (0.51 g; 0.46
mmol) in H.sub.2O (50 mL) at 20.degree. C. (pH 2.93); after each
addition the suspension was stirred until complete dissolution.
Once the complexation was complete the pH was adjusted to 7 with
0.1 N NaOH and the solution was desalted by elution with
water/acetone from a column of Amberlite.RTM. XAD-1600 resin (100
mL; diam. 3 cm). The fractions containing the desired product and
free from salts were collected and evaporated to give the compound
of Formula IV (0.37 g, 0.28 mmol) a yellow solid. Yield: 61%.
[0153] The immunoassay method was designed according to: Hemmila I
et al. Anal Biochem. 1984 March; 137(2): 335-43. The solutions used
were as described in the Example 4 with the following exceptions: a
DELFIA.RTM. Wash (Perkin Elmer) was used instead of the above
Washing buffer. The Tracer stock solution contained the Europium
100 .mu.M and it was stored at +4.degree. C., protected from light.
Just before use it was diluted 1:300000 in Assay Buffer to obtain a
final concentration of 440 .mu.g/mL.
[0154] The assay was performed in DELFIA.RTM. Yellow plates (Perkin
Elmer). After the 3-h incubation with the mixed samples, the
Formula V diluted compound solution was added (50 .mu.L per well)
to all wells. The plates were then sealed with plastic adhesive
sheets and incubated under agitation for 1 h at 37.degree. C.
[0155] After three washes, the plates were tapped dry on absorbent
paper, and Delfia Enhancement Solution (Perkin Elmer) was added
(200 .mu.L) After 1 h at 25.degree. C., the plates were read in a
Victor3 instrument according to the "Europium" manufacturer
protocol.
[0156] A calibration curve was prepared using nine concentrations
of the test item in the range 30-2000 .mu.g/mL. The curve is shown
in FIG. 1, panel b).
Example 6
Synthesis of HRP-T3S Monoamide
[0157] The conjugate was prepared directly using a commercial kit
containing activated HRP (e.g., HOOK.TM. HRP PLUS Labeling
Kit-G-Biosciences). 1-2 mg of T3S was dissolved in 1 mL of the
supplied carbonate buffer, then this solution was dispensed in a
vial containing the lyophilized activated HRP, mixing gently by
repeated pipetting in order to reconstitute the activated enzyme.
After about 1 hour at room temperature, 20 .mu.L of the supplied
Sodium Cyanoborohydride (NaCNBH3) solution was added, then allowing
to react for about 15 min at room temperature. Finally, 50 .mu.L of
quenching buffer was added, then incubating with gentle tumbling or
shaking for 15 min. The final conjugate was desalted and buffer
exchanged in PBS by either dialysis or column desalting.
Appropriate dilutions of the T3S-HRP conjugate were prepared and
used as a tracer in a single-step ELISA, as described in Example 4,
omitting the Streptavidin-HRP incubation step.
##STR00011##
Comparative Example
T3S Synthesis in DMF and Elution Trials
##STR00012##
[0159] The reaction was carried out according to the scheme above,
in DMF. Briefly: T3 (40 mg) was dissolved in ammoniacal ethanol.
This solution was evaporated under a stream of nitrogen.
[0160] To the residual, 2 ml of a hot solution of Chlorosulfonic
acid (obtained by mixing 2.5 mL of 99% Chlorosulfonic acid and 8 ml
of N,N-DMF) was added. Subsequently, the mixture was allowed to
reach room temperature under stirring and the reaction was
continued overnight.
[0161] The mixture was diluted with water (5 mL) and then was
eluted on a column of Sephadex LH-20 (5 mL), obtaining fraction A.
The elution was continued with 0.1 N HCl (5 mL), obtaining fraction
B.
[0162] These fractions were re-loaded on column and purified by
serial elution of 0.1 N HCl (approx. 4 mL), water and absolute
Ethanol. However, five different water and absolute ethanol
quantities were used for purification. The T3S yields and purities
obtained by these five conditions have been summarized In Table
B.
TABLE-US-00006 TABLE B Purification trials T3-Sulfate from
T3-Sulfate from aqueous alcoholic fractions abs. fractions H.sub.2O
EtOH Amount Purity.sup.(a) Yield Amount Purity.sup.(a)( b)
Yield.sup.(c) Trial (mL) (mL) (mg) (%) (%) (mg) (%) (%) 1 5 10 1.0
100 2.2 35 80 62.3 2 50 100 2.5 100 5.6 30 80 53.4 3 125 125 8.0
100 17.8 30 75 50.1 4 Not 100 Not Not -- 30 75 50.1 registered
registered registered 5 40 10 Not Not -- 10 50 11.1 registered
registered 20 Not Not -- 20 70 31.2 registered registered .sup.(a)
1H-NMR purity. .sup.(b) From the analyses, the product is a mixture
of T3S and T3. .sup.(c) Yields are calculated on the content of
T3-Sulfate.
[0163] Table B shows that when the synthesis is carried out in the
conditions described above and DMF is used as the solvent, high
conversion may be achieved, but the overall yield is quite low.
* * * * *