The invention relates to cyclodextrins and derivatised cyclodextrins, such as
sulphoalkyl ether b-cyclodextrin, and in particular to a novel method for the synthesis
thereof. The invention is particularly concerned with producing sulphobutyl ether b-
cyclodextrin. The invention extends to novel compositions comprising sulphoalkyl
ether b-cyclodextrins, and to the uses of such compositions, for example as excipients
in order to improve the solubility and chemical stability of drugs in solution.
Sulphobutyl ether b-cyclodextrin (SBE^-CD or SBECD) is one of a class of polyanionic,
hydrophilic water soluble cyclodextrin derivatives. The parent b-cyclodextrin can form
an inclusion complex with certain active pharmaceutical ingredients (API) with two
benefits, the apparent aqueous solubility of the API increases and, if labile functional
groups are included, chemical stability is improved. However, the parent b-cyclodextrin
suffers from two problems, including lower aqueous solubility and nephrotoxicity when
given via injection, e.g. the intravenous route. Derivatisation of b-cyclodextrin (and its
variants a and g -cyclodextrin) has been shown to be beneficial with respect to both of
these two defects. The first derivatised cyclodextrin was the hydroxypropyl derivative,
which was later followed by sulphobutyl ether. These two derivatised cyclodextrins are
the most commercially significant.
Figure l illustrates the chemical reaction for the synthesis of SBE^-CD from the
reagents b-cyclodextrin (b-CD) and 1, 4-butane sultone (BS). US 6,153,746 (Shah et al,
2000) describes a batch synthesis of SBE^-CD, the process being effectively divided
into three main stages, i.e. initial reagent dissolution, a sulphoalkylation reaction and
final reaction quenching. The reaction is then followed by downstream processing and
purification, and ultimate isolation of the solid SBE^-CD material. However, a
problem associated with using a batch synthetic method is that a high proportion of
lower substituted SBE^-CD is observed. There is therefore a need to provide an
improved synthetic method for producing substituted cyclodextrins, such as SBE^-CD.
SBE^-CD is currently used as an effective pharmaceutical excipient, and has been
given the trade name Captisol (RTM). To date, there are five US FDA-approved, SBE-b-
CD-enabled drug products on the market: Nexterone (Baxter International); Geodon
(Pfizer); Cerenia (Zoetis); Kyprolis (Onyx); Abilify (Bristol Myers Squibb).
In addition, as described in US 6,632,80361 (Harding, 2003), Pfizer has developed the
clinically important antifungal drug, voriconazole, formulated with SBE- -CD, as
excipient. If Shah's and Harding's patents are considered together, the overall process
to produce an injectable form of voriconazole follows a 10-step scheme, as shown in
Figure 22. Production of the SBE- -CD excipient is represented by the six white boxes,
and production of the final injectable voriconazole (i.e. formulation of the API with the
excipient) is represented by the four grey boxes. The problems with this process are
that it includes many steps, one of which is the transportation of the SBE- -CD from
the fine chemical manufacturing plant to the customer who adds the active ingredient,
e.g. voriconazole. Furthermore, freeze drying and spray drying are expensive and timeconsuming
processes. There is therefore a need to provide an improved process for the
production of pharmaceuticals comprising substituted cyclodextrin-based excipients.
As described in the Examples, the inventors carefully studied the batch SBE- -CD
production method that is described in US 5,376,645 (Stella et al, 1994), and
experimented with the stoichiometry of the reaction, and have devised a significantly
improved continuous flow synthetic method for producing sulphoalkyl ether
cyclodextrins, such as SBE- -CD.
Hence, according to a first aspect of the invention, there is provided a method for
preparing sulphoalkyl ether b-cyclodextrin, the method comprising contacting
cyclodextrin with a base to form activated cyclodextrin, and separately contacting the
activated cyclodextrin with an alkyl sultone to form sulphoalkyl ether b-cyclodextrin,
characterised in that the sulphoalkylation reaction is carried out under continuous flow
conditions.
In a second aspect, there is provided sulphoalkyl ether b-cyclodextrin obtained or
obtainable by the method according to the first aspect.
The inventors have found that the continuous flow nature of the sulphoalkylation
reaction in the method of the first aspect results in a surprisingly superior process
compared to the prior art batch process, because it exhibits a greater reaction efficiency
and results in a much tighter control of substitution of the resultant sulphoalkyl ether
b-cyclodextrin, which is preferably sulphobutyl ether b-cyclodextrin (i.e. SBE^-CD).
Indeed, the continuous flow synthesis process of the invention substantially less than
50% of the amount of base (which is preferably sodium hydroxide) that is used in the
prior art batch process, and only a 7:1 molar ratio of the alkyl sultone (which is
preferably, 1, 4-butane sultone) to cyclodextrin instead of the 10:1 used by the prior art
method. This finding was completely unexpected, since the inventors' expectation was
that, at best, an equivalent synthetic efficiency between the batch and continuous flow
methods would be seen. Accordingly, by using the continuous flow method of the first
aspect, the alkyl sultone can react with the cyclodextrin more efficiently and completely
to thereby generate higher degrees of substitution with more efficient use of the starting
materials. It has also been noted that lower volumes of water are necessary to achieve
chemical coupling.
In one embodiment of the method of the invention, the sulphoalkylation reaction is
carried out under continuous flow conditions, whereas the activation reaction may be
carried out either continuously, batch, or fed-batch. Preferably, however, the activation
reaction is carried out as a batch process while the sulphoalkylation reaction is carried
out under continuous flow conditions.
Problems associated with prior art methods which are fully batch, or fully continuous,
(i.e. with respect to both the activation stage and the sulphoalkylation reaction stage)
are that they result in the production of high concentrations of by-products (e.g.
dimerisation products), produce SBE- -CD with low average degrees of substitution, as
well leave unreacted alkyl sultone. Accordingly, the method of the invention, in which
the activation stage is batch and the sulphoalkylation reaction stage is continuous,
results in lower concentrations of by-products, SBE- -CD with a higher average degree
of substitution, and also most if not all of the alkyl sultone is reacted.
Preferably, the cyclodextrin is a-, b- or g -cyclodextrin. It will be appreciated that a - and
g -cyclodextrin can be used as pharmaceutical excipients for instance in commercially
available drugs such as Prostavasin, Opalamon (b-CD) and Voltaren (modified g -CD).
Most preferably, however, the cyclodextrin is b-cyclodextrin.
The alkyl sultone may comprise propane sultone. Thus, sulphoalkyl ether b-
cyclodextrin preferably comprises sulphopropyl ether b-cyclodextrin ( RE-b- ) .
However, most preferably the alkyl sultone comprises 1, 4-butane sultone. Preferably,
therefore, the sulphoalkyl ether b-cyclodextrin comprises sulphobutyl ether b-
cyclodextrin (SBE^-CD).
The resultant substituted sulphoalkyl ether b-cyclodextrin according to the second
aspect is novel per se because it exhibits a higher average degree of substitution, for a
lower input of alkyl sultone and base than that which is produced using the known
batch process. The batch method of preparing substituted sulphoalkyl ether b-
cyclodextrin produces a higher concentration of lower degrees of sulphoalkyl ether b-
cyclodextrin substitution than that produced using continuous flow. As shown in
Figures 20 and 25, the continuous flow process of the invention however results in a
lower concentration of lower substituted sulphoalkyl ether b-cyclodextrin (i.e. a degree
of substitution value of 1-4) and surprisingly much higher concentrations of the higher
substituted sulphoalkyl ether b- cyclodextrin (i.e. individual degrees of substitution
values of 4-13).
Thus, preferably the average degree of substitution (ADS) of the sulphoalkyl ether b-
cyclodextrin produced by the method of the first aspect or the SBE^-CD of the second
aspect is greater than 7, more preferably 7.3 or more, more preferably 8 or more, even
more preferably 9 or more, and most preferably 10 or more. The skilled person will
appreciate that it is possible to calculate the substitution degree (i.e. the substitution
envelope) by using the following Formula:
ADS= å ((PAC)x (MT)/SCAx ioo)/ioo
where PACrefers to the peak area count; MT refers to the migration time; and SCA
refers to the summation of corrected area. The inventors believe that this increased
ADS is an important feature of the invention.
Thus, in a third aspect there is provided a composition comprising sulphobutyl ether b-
cyclodextrin (SBE^-CD), wherein the average degree of substitution (ADS) is 7 or
more, preferably 7.3 or more, preferably 8 or more, even more preferably 9 or more,
and most preferably 10 or more.
Since the batch method produces a higher proportion of lower substituted SBE^-CD
than higher substituted SBE^-CD, the continuous flow method of the invention
provides a significant advantage.
Preferably, the composition of the third aspect comprises SBE- -CD having a
Substitution Molecular Mass Fraction (SMF) greater than 0.57, more preferably greater
than 0.58, and even more preferably greater than 0.59. Preferably, the composition of
the third aspect comprises SBE- -CD having an SMF greater than 0.60, and more
preferably greater than 0.61. Example 8 describes how the SMF value can be calculated
with reference to Figure 28.
The base may be an alkali metal hydroxide, for example sodium hydroxide, lithium
hydroxide or potassium hydroxide. It is preferred that the base comprises sodium
hydroxide.
The molar ratio of base (which is preferably sodium hydroxide) to cyclodextrin is
preferably within the range of 2:1 to 22:1, preferably 6:1 to 20:1, more preferably 6:1 to
15:1, and even more preferably 6:1 to 14:1. The preferred molar ratio of base to
cyclodextrin is 6:1 to 14:1. The most preferred molar ratio of base to cyclodextrin is 6:1
to 15:1.
During their research, the inventors carefully considered the prior art batch process,
and found that the base, employed to chemically activate the b-cyclodextrin hydroxyl
groups, has a tendency to attack the alkyl sultone reagent, thereby reducing its effective
concentration, and, as a result, reduces the average degree of substitution in the final
product with the generation of low degree of substitution species. Accordingly, during
the method of the first aspect, it is preferred that the base is kept separate from the
alkyl sultone, preferably 1, 4-butane sultone. Preferably, the base is first separately
reacted with the cyclodextrin in order to produce the activated cyclodextrin. This
reaction is preferably conducted in a first reservoir vessel. The activation reaction may
therefore be carried out as a batch or fed-batch process. Preferably, the base and
cyclodextrin form an aqueous solution. The activation reaction is preferably conducted
at a temperature of about 50 to 95 °C, more preferably 60 to 70°C. The activation
reaction is preferably conducted at atmospheric pressure.
Preferably, the alkyl sultone is contained within a second reservoir vessel. Preferably,
the first and second vessels are not directly connected to each other, such that the
sultone and the base do not react with each other.
The activated cyclodextrin (i.e. aqueous solution) and the alkyl sultone (i.e. pure) are
preferably fed to a confluent 3-way junction where they are allowed to react to produce
the substituted sulphoalkyl ether b-cyclodextrin. The activated aqueous cyclodextrin
and the alkyl sultone are preferably pumped at a controlled rate to the junction.
The molar ratio of sultone (preferably 1, 4-butane sultone) to cyclodextrin (preferably
b-cyclodextrin) is preferably between about 7:1 and 33:1. Preferably, the molar ratio of
sultone to cyclodextrin is 7:1 to 17:1.
The sulphoalkylation reaction is preferably conducted at a temperature of 60 to ioo°C,
more preferably 65 to 95 °C, and even more preferably 60 to 70°C. The
sulphoalkylation reaction is preferably conducted at atmospheric pressure.
The alkylation reaction may be carried out in a continuous stirred tank reactor (CSTR)
or a flow reactor with efficient mixing and of suitable length to allow the reaction to
complete within the reactor tubing. The activation of b-cyclodextrin is an important
process parameter prior to reaction and this must continue irrespective of the reactor
architecture.
In a preferred embodiment, the method of the invention comprises contacting the
cyclodextrin in a batch or fed-batch reaction with the base to form activated
cyclodextrin, and separately contacting the activated cyclodextrin with an alkyl sultone
to form sulphoalkyl ether b-cyclodextrin, wherein the sulphoalkylation reaction is
carried out under continuous flow conditions.
In a most preferred embodiment, therefore, the method comprises separately reacting
b-cyclodextrin with sodium hydroxide in a batch or fed-batch reaction to form activated
b-cyclodextrin, and then separately contacting the activated b-cyclodextrin with 1, 4-
butane sultone to form SBE^-CD under continuous flow conditions.
The inventors have surprisingly demonstrated that it is possible to accurately control
and manipulate the average degree of substitution (ADS) of sulphoalkyl ether b-
cyclodextrin produced in sulphoalkylation reaction by varying the sodium hydroxide
concentration in the initial activation reaction.
Preferably, therefore, the method comprises controlling the average degree of
substitution (ADS) of sulphoalkyl ether b-cyclodextrin in the sulphoalkylation reaction
by varying the base concentration in the initial activation reaction. This is an important
feature of the invention.
Accordingly, in another aspect, there is provided use of sodium hydroxide
concentration for controlling the average degree of substitution (ADS) of sulphoalkyl
ether b-cyclodextrin produced in a sulphoalkylation reaction between activated
cyclodextrin and an alkyl sultone.
Preferably, the use comprises carrying out an initial activation reaction between
cyclodextrin and the base to form activated cyclodextrin.
To date, no one has appreciated that the concentration of sodium hydroxide can be
varied in the initial activation reaction in order to control and manipulate the average
degree of substitution (ADS) of resultant sulphoalkyl ether b-cyclodextrin.
The unsubstituted parent b-CD is shown to induce irreversible nephrotic damage to the
kidney cells when used as an excipient in injection formulations. SBE^-CD causes
reversible vacuolation of renal cells but not nephrotic damage and is therefore
preferred for use in injectable formulations. Given that the inventors have clearly
demonstrated that the method of the invention results in a lower concentration of low
degree of substitution SBE^-CD species it is believed that the SBE^-CD may cause
lower levels of physiological changes to renal cells. Accordingly, they believe that the
SBE^-CD of the second aspect or the composition of the third aspect can be used to
reduce changes in renal cells when used as a drug delivery system.
Hence, in a fourth aspect there is provided the use of sulphoalkyl ether b-cyclodextrin
of the second aspect, or the composition of the third aspect, as a drug delivery system.
Preferably, the drug delivery system is an excipient, which preferably exhibits little or
no side effects with regard to renal physiology. Preferably, the sulphoalkyl ether b-
cyclodextrin comprises sulphobutyl ether b-cyclodextrin ( BE-b- ).
In a fifth aspect, therefore, there is provided a pharmaceutical excipient comprising the
sulphoalkyl ether b-cyclodextrin of the second aspect, or the composition of the third
aspect.
Preferably, therefore, the sulphoalkyl ether- b-cyclodextrin comprises sulphobutyl ether
b-cyclodextrin (SBE^-CD).
Advantageously, as described in examples, use of the continuous flow method of
invention means that it is now possible to combine the two processes shown in Figure
22 (i.e. excipient production, and pharmaceutical production), to result in the 6-step
process chain shown in Figure 23.
Hence, in a sixth aspect, there is provided a method of preparing a pharmaceutical
composition, the method comprising preparing the pharmaceutical excipient according
to the fifth aspect, and contacting the excipient with an active pharmaceutical
ingredient (API) to produce a pharmaceutical composition.
In contrast to the process shown in Figure 22, the production of the sulphobutyl ether
cyclodextrin, acting as excipient, is now represented in Figure 23 byjust three white
boxes (instead of six), and formulation of the API with the excipient to create the
pharmaceutical product is represented by only three grey boxes (instead of four).
Accordingly, the method of the invention means that three of the steps shown in Figure
22 can be omitted. Therefore, it is now unnecessary to transport the sulphobutyl ether
cyclodextrin from the fine chemical manufacturer to the customer. This would also
include warehousing, etc. Secondly, the sulphoalkyl ether b-cyclodextrin can be
manufactured on a just-in-time, just-enough basis. Thirdly, one of the two expensive
and time-consuming freeze or spray drying process steps can be avoided.
Preferably, the method comprises contacting the excipient with an active
pharmaceutical ingredient (API) to produce a pharmaceutical composition without
drying or isolating the excipient.
Preferably, the pharmaceutical excipient comprises sulphobutyl ether b-cyclodextrin.
Preferably, the active pharmaceutical ingredient comprises voriconazole, ziprasidone,
aripiprazole, maropitant, amiodarone, or carfilzomib, or their salts, solvates,
polymorphs, pseudopolymorphs or co-crystals.
In another embodiment, the method of the invention comprises separately reacting b-
cyclodextrin with sodium hydroxide to form activated b-
cyclodextrin, and then separately contacting the activated b-cyclodextrin with 1, 4-
butane sultone to form SBE- -CD, all under continuous flow conditions.
Advantageously, the stoichiometry of the reaction can be readily controlled by varying
the flow rates of the activated cyclodextrin solution and/ or liquid sultone.
Hence, in another aspect of the invention, there is provided a method for preparing
sulphoalkyl ether b-cyclodextrin, the method comprising contacting cyclodextrin with a
base to form activated cyclodextrin, and separately contacting the activated
cyclodextrin with an alkyl sultone to form sulphoalkyl ether b-cyclodextrin,
characterised in that the process is carried out under continuous flow conditions.
All of the features described herein (including accompanying claims, abstract and
drawings), and/ or all of the steps of any method or process so disclosed, may be
combined with any of the above aspects in any combination, except combinations
where at least some of such features and/ or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same
may be carried into effect, reference will now be made, by way of example, to the
accompanying drawings, in which: -
Figure 1shows the chemical reaction for the synthesis of sulphobutyl ether b-
cyclodextrin (SBE^-CD) from b-cyclodextrin (CD) and 1, 4-butane sultone (BS);
Figure 2 is a schematic representation for an embodiment of an apparatus for carrying
out continuous flow (CF) synthesis for SBE^-CD according to the invention;
Figure 3 shows the actual lab-based apparatus for carrying out a continuous flow
synthesis for SBE^-CD;
Figure 4 is a graph showing the changing amount of NaOH at 7:1 and 10:1 BS/CD
mole ratio; 100% nominal sodium hydroxide is equivalent to the base content used in
US 5,376,645 (Stella et 994)
Figure 5 shows electropherograms of batch manufactured SBE^-CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE^-CD manufactured by a continuous flow
process according to the invention, with a 8:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 6 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 11:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 7 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 14:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 8 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 17:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 9 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 19:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 10 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 23:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 11 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 28:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 12 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 33:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 11:1.
Figure 13 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according to the invention, with a 7:1 butane sultone to b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide to b-CD molar ratio is 6:1.
Figure 14 shows electropherograms of batch manufactured SBE- -CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE- -CD manufactured by a continuous flow
process according t o the invention, with a 7:1 butane sultone t o b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide t o b-CD molar ratio is 9:1.
Figure 15 shows electropherograms of batch manufactured SBE -P-CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE -P-CD manufactured by a continuous flow
process according t o the invention, with a 7:1 butane sultone t o b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide t o b-CD molar ratio is 11:1.
Figure 16 shows electropherograms of batch manufactured SBE -P-CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE -P-CD manufactured by a continuous flow
process according t o the invention, with a 7:1 butane sultone t o b-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide t o b-CD molar ratio is 14:1.
Figure 17 shows electropherograms of batch manufactured SBE -P-CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE -P-CD manufactured by a continuous flow
process according t o the invention, with a 10:1 butane sultone t o P-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide t o P-CD molar ratio is 6:1.
Figure 18 shows electropherograms of batch manufactured SBE -P-CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE -P-CD manufactured by a continuous flow
process according t o the invention, with a 10:1 butane sultone t o P-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide t o P-CD molar ratio is 9:1.
Figure 19 shows electropherograms of batch manufactured SBE -P-CD (US 6,153,746-
Shah et al, 2000) as the solid line and SBE -P-CD manufactured by a continuous flow
process according t o the invention, with a 10:1 butane sultone t o P-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide t o P-CD molar ratio is 11:1.
Figure 20 shows electropherograms of batch manufactured SBE -P-CD (US 6,153,746-
Shah, et al 2000) as the solid line and SBE -P-CD manufactured by a continuous flow
process according t o the invention, with a 10:1 butane sultone t o P-cyclodextrin molar
ratio as the dotted line. The sodium hydroxide t o P-CD molar ratio is 14:1.
Figure 21 shows a Vapourtec integrated flow reactor and associated equipment that
will ultimately b e preferred for the integrated manufacture of the SBE -P-CD and API in
a secondary pharmaceutical production manufacturing area t o produce the drug
product.
Figure 22 is a schematic representation of a conventional process chain for
voriconazole Injection Based on the standard SBECD batch process and Fine Chemical
Model (excipient production in white; secondary pharmaceutical production in grey).
Figure 23 is a schematic representation of a revised process chain for voriconazole
Injection Based on an SBECD continuous flow (CF) process and Integrated
Manufacture Model (excipient production in white; secondary pharmaceutical
production in grey).
Figure 24 is a chromatogram of sulphobutylether b-cyclodextrin produced by the
method described in US 6,153,746 (Shah, 2000), and tested according to the
methods described in United States Pharmacopoeia 35/National Formulary 30
(USP35/NF30). HPLC conditions are based on a gradient separation with a CDScreen-
DAP column and ELSD detection.
Figure 25 is a chromatogram of sulphobutylether b-cyclodextrin produced by the
method according to the invention. Reaction conditions correspond to those used to
generate Figure 20, and HPLC conditions are based on a gradient separation with a
CD-Screen-DAP column and ELSD detection.
Figure 26 is a table showing shows a summary of the data adding the Average
Degree of Substitution data and dispersion data.
Figure 27 is a table describing an attempt to produce material compliant with the
USP35/NF30 monograph with the use of more moderate reaction conditions.
Figure 28 is a graph showing substitution molecular mass ratio for SBE- -CD.
Figure 29 is a graph showing the molecular weight and individual degree of
substitution for SBE^-CD.
Example
The inventors have developed a novel continuous flow (CF) method for the synthesis of
sulphoalkyl ether- b-cyclodextrin, for example sulphobutyl ether b-cyclodextrin (SBE-b-
CD). The invention includes novel compositions comprising sulphoalkyl ether b-
cyclodextrins, and to therapeutic uses of such compositions, for example to improve the
solubility and chemical stability of drugs in solution.
Materials
Beta cyclodextrin (b-CD), 1, 4-Butane Sultone (BS), Water for injections and sodium
hydroxide (NaOH).
Laboratory Equipment
Continuous stirred tank reactor (CSTR) vessel, Masterflex pump, Hotplate stirrer,
Water bath, PTFE tubing (2mm ID/4mm ID), Omnifit Connectors, Dialysis tubing
(Biotech grade, Cellulose Ester, 0.5-1 kDa MWCO).
Methods
The set-up for the continuous flow experiments consisted of two Masterflex pumps (8,
10) connected to a double 10ml (i.e. two 10ml chambers) jacketed continuous stirred
tank reactor (CSTR) or holding chamber (14) used as a holding chamber/sight glass.
The two pumps (8, 10) were connected to the CSTR/holding chamber (14) via a threeway
connector (12) and PTFE tubing. Non-return valves were fitted in line in the
vicinity of the three-way connector (12) to prevent the reagent stream reverse flow as a
result of differential flow pressure in either of the feed lines. In one embodiment, the
PTFE tubing was put in a water bath to maintain temperature at approximately 50-
6o°C. In another embodiment, the PTFE tubing was put in a water bath to maintain
temperature at approximately 6o-ioo°C.
In a round bottom flask, a stock solution of b-CD in NaOH solution (4) was first
prepared as follows: I5g of b-CD (1.32 x io 2 mole) was added with stirring to an
aqueous solution composed of 6g ofNaOH in 30ml water. This solution was
maintained between 6o-70°C with a hotplate stirrer.
At the given drive speeds, pump (8) was used to deliver stock b-CD solution into the
CSTR (14) via a three way connector (12) where the reaction initially takes place, while
pump (10) was used to also deliver neat butane sultone (6), at ambient temperature,
through the connector (12) into the CSTR (14). However, in some embodiments, the
neat sultone (6) can be heated to 60-90 °C. The CSTR (14) contained two 10ml
chambers and was provided to increase the residence time for the reaction to continue,
having started in the connector (12). In one embodiment, pump (8) was first turned on
to feed the b-CD until it reached the first chamber of the CSTR (14), after which pump
(10) was then turned on to feed the butane sultone into the CSTR (14). However, in
another embodiment, pumps (8, 10) are both activated at the same time in order to
avoid pumping pure b-CD through the system to produce higher than desirable
unreacted precursor that would ultimately need to be removed by downstream
processing. An internal vortex circulation was generated within the continuous flowing
reaction stream within the CSTR (14), which ensured rapid mixing. Efficient stirring
appears to be very important to the success of the process. The reaction solution was
delivered via pumps (8, 10) into the CSTR (14) in a continuous manner.
The PTFE tubing is about 30cm in length and is not sufficient for the reaction to
complete prior to entry into the CSTR (14). As two phases are seen in the first chamber
of the CSTR (14), it is most likely that small volumes of the heated reagents are
delivered and react there. Provided that the flow rate is not excessively high, the
second chamber of the CSTR (14) and the receiving vessel both contain clear liquid
suggesting that the reaction is complete upon exit from the first chamber of the CSTR
(14). High flow rates will deliver unreacted material to the second chamber and, in
extreme circumstances, to the receiving vessel. The crude product was harvested in a
20ml sample bottle.
Continuous flow experiments were carried out at different drive speed combinations for
pump (8) and (10) thus obtaining a series of BS:CD mole ratio, as shown in Tables 1
and 2.
Table 1 - The relationship between pump drive speed and flow rate giving rise to
different butane sultone- b-cyclodextrin molar ratios - constant b-cyclodextrin flow
rate.
Table 2 - Tΐ e relationship between pump drive speed and flow rate giving rise to
different butane sultone- b-cyclodextrin molar ratios - constant butane sultone flow
rate.
b-CD BS
Drive 11 15 5
speed(rpm)
Flow 0.99 1-35 0.45
rate(ml/min)
Concentration 4-36 5-94 4.4 X l O 3
- i5 -
5
In addition, the effect of changing the amount ofNaOH at a given drive speed/BS^-CD
mole ratio was also carried out, thus obtaining a series of NaOH: CD mole ratios, as
shown in Figure 4. The crude reaction products were dialysed and lyophilized to obtain
the sulphobutyl ether of b-CD as a white solid intermediate for chemical analysis. The
product was analysed using capillary electrophoresis, mass spectrometry and HPLC to
show the degree of substitution, HPLC was carried out to show unreacted b-CD and
levels of BS residues were analysed by gas chromatography. The lyophilised product
was weighed to give the yield.
Example 1
Referring to Figures 2 and 3, there are shown embodiments of the apparatus 2 for the
continuous flow synthesis of SBE-P-CD. Two reservoirs (4), (6) are primed, the first
reservoir (4) containing "activated" b-cyclodextrin and sodium hydroxide in an aqueous
solution, and the second reservoir (6) containing pure 1,4-butane sultone. Afirst
peristaltic pump (8) was turned on to feed the b-cyclodextrin and sodium hydroxide in
aqueous solution through a three-wayjunction (12) with non-return valves. A second
peristaltic pump (10) was turned on to feed the 1, 4-butane sultone also through the
junction (12) where it reacted with the b-cyclodextrin. The stoichiometry of the
reaction could be controlled by mixing the two reaction streams at differential rates,
and the ratio of b-cyclodextrin to sodium hydroxide could be adjusted in the reservoir
(4) prior to mixing with 1, 4-butane sultone. The amount of SBE^-CD produced in the
process is therefore a function of pumping time, and not equipment scale. The
residence time of the reaction between the b-cyclodextrin/NaOH solution and 1, 4-
butane sultone was increased by passing the mixture from the outlet of the three-way
junction (12) to a holding chamber/ sight glass or continuous stirred tank reactor
(CSTR) (14) where further reaction took place. The CSTR (14) could be replaced in
whole or in part with a temperature controlled coiled tubing of sufficient length. This
would provide appropriate level of turbulence and residence time for the coupling
reaction to complete efficiently.
The inventors' primary focus was to study the complexity of the sulphoalkylation
reaction in the flow synthesis mode. It was therefore necessary to dialyse (18) the
reaction effluent (16), freeze dry it (20) and then analyse it (22). Under commercial
conditions, the SBE^-CD effluent (16) leaving the CSTR (14) would be connected to the
downstream processing elements, e.g. continuous dialysis, flow-through
depyrogenation columns and membrane pre-frlters (pore size 0.22 mp or greater) prior
to dynamic active pharmaceutical ingredient addition processes described in Figure 23.
The SBE- -CD produced has been analysed by mass spectrometry, capillary
electrophoresis for comparison with the patent literature. The other processing simply
becomes an engineering problem as it involves mixing or purification of aqueous SBE-
b-CD or SBE- -CD-drug complex solutions.
Results
Referring to Figures 5-20, there are shown electropherograms for the standard batch
(standard) and continuous flow (CF) synthesis of SBE- -CD at the different BS: b-CD
molar ratios resulting from differential pump speeds at a constant b-CD: sodium
hydroxide mass ratio, or at different b-CD: sodium hydroxide mass ratios for two
different BS: b-CD molar ratios as indicated. The standard curve (solid line)
corresponds to the known batch manufacture method of SBE^-CD, as described in US
6,153,746 (Shah et al, 2000). The dotted trace in each graph however is for an SBE-b-
CD sample produced by a continuous flow (CF) synthesis process according to the
invention.
Referring to Figure 5, there is shown the electropherograms for SBE^-CD produced
using the known batch method compared to continuous flow (CF) with a 1, 4-Butane
Sultone (BS): Beta cyclodextrin (b-CD) drive speed of 3:8 and with a given BS: CD mole
ratio as shown in Table 1. As can be seen, there are 10 peaks for the CF method and only
9 peaks for the Batch method. The number of peaks is indicative of the degree of
substitution for the derivatives.
Referring to Figure 6, there is shown the electropherograms for standard sample of
Batch produced SBE^-CD and SBE^-CD produced by continuous flow synthesis at a
4:8 BS/CD drive speed and therefore a given BS: CD mole ratio as shown in Table 1. As
can be seen, there are 10 peaks for the CF method and only 9 peaks for the Batch
method. The number of peaks is indicative of the degree of substitution for the
derivatives.
Referring to Figure 7, there is shown the electropherograms for standard sample of
Batch produced SBE-P-CD and SBE-P-CD produced by continuous flow synthesis at a
5:8 BS/CD drive speed, at a given BS: CD mole ratio as shown in Table 1. The two
electropherograms show coincidence which indicates equivalence of substitution
envelope. Both plots show about 9 distinguishable peaks which correspond to the
degree of substitution.
Referring to Figure 8, there is shown the electropherograms for standard sample of
Batch produced SBE-P-CD and SBE-P-CD produced by continuous flow synthesis at a
6:8 BS/CD drive speed at a given BS: CD mole ratio as shown in Table 1. The two
electropherograms show coincidence which indicates equivalence of substitution
envelope. Both plots show about 9 distinguishable peaks which correspond to the
degree of substitution.
Referring to Figure 9, there is shown the electropherograms for standard sample of
Batch produced SBE-P-CD and SBE-P-CD produced by continuous flow synthesis at a
7:8 BS/CD drive speed and at given BS: CD mole ratio as shown in Table 1. The
electropherogram for the continuous flow shows an intense peak between 4 and 5
minutes, this peak possibly indicating the presence of a reaction impurity. The BS: PCD
mole ratio indicates an excess of BS.
Referring to Figure 10, there is shown the electropherograms for standard sample of
Batch produced SBE-P-CD and SBE-P-CD produced by continuous flow synthesis at a
8:8 BS/ P-CD drive speed at given BS: CD mole ratio as shown in Table 1. As can be
seen, there are 10 peaks for the CF method and only 9 peaks for the Batch method. The
number of peaks is indicative of the degree of substitution for the derivatives.
Referring to Figure 11, there is shown the electropherograms for standard sample of
Batch produced SBE-P-CD and SBE-P-CD produced by continuous flow synthesis at a
10:8 BS/CD drive speed at given BS: CD mole ratio as shown in Table 1. The
electropherogram for the continuous flow shows an intense peak between 4 and 5
minutes, again this peak possibly indicating the presence of a reaction impurity. The
BS: P-CD mole ratio indicates an excess of BS.
Referring to Figure 12, there is shown the electropherograms for standard sample of
Batch produced SBE-P-CD and SBE-P-CD produced by continuous flow synthesis at a
12:8 BS/CD drive speed at given BS: CD mole ratio as shown in Table 1. The
electropherogram for the continuous flow also shows an intense peak between 4 and 5
minutes, this peak possibly indicating the presence of a reaction impurity. The BS: b -
CD mole ratio indicates an excess of BS.
Referring to Figure 13, there is shown the electropherograms batch manufactured SBEP-
CD as the solid line and SBE-P-CD manufactured by the continuous flow process with
a 7:1 butane sultone to b-cyclodextrin molar ratio as the dotted line. The sodium
hydroxide to b-CD molar ratio is 6:1. As can be seen, coincidence of the two
electropherograms indicates an equivalent 'Substitution Envelope', i.e. degree of
substitution distribution. However, it is remarkable that the continuous flow synthesis
process of the invention requires less than 50% of the sodium hydroxide that is used in
the prior art batch process (Stella et al, 1994), and a 7:1 molar ratio of 1, 4-butane
sultone to b-cyclodextrin instead of the 10:1 used by the prior art method. This finding
was completely unexpected, given that the inventors' expectation was at best an
equivalent synthetic efficiency. Although the inventors do not wish to be bound by any
theory, it would appear that the shielding of sodium hydroxide from 1,4-butane sultone
up to the point where the reaction streams mix and the reaction takes place allows for
an efficient activation of b-cyclodextrin hydroxyl groups at the point of the reaction
with minimal degradation of 1,4-butane sultone to low molecular weight by-products.
In short, using the continuous flow method of the invention, 1,4-butane sultone can
react with b-cyclodextrin more efficiently and completely to generate higher degrees of
substitution with more efficient use of the starting materials.
The average degree of substitution (ADS) can be readily determined using the following
formula taken from US7, 635,7762 (Antle, 2009):-
ADS= å ((PAC)x (MT)/SCA x ioo)/ioo
Where PACrefers to the peak area count; MT refers to the migration time; and SCA
refers to the summation of corrected area.
To test this hypothesis further, the inventors attempted to increase the ratio of sodium
hydroxide to b-cyclodextrin, and the results are shown in Figures 14-20. In the prior art
batch process, according to Shah, this would have no beneficial effect on the average
degree of substitution or the distribution of low degree of substitution species, i.e. a
change in the substitution envelope, because the sodium hydroxide would simply
destroy the 1,4-butane sultone before reaction with the hydroxyls could take place. In
essence, there is a kinetic limit to the degree of substitution under batch processing
conditions. Shah exploits this to maximise the degree of substitution and reduce the
residual concentration of reactants.
Referring to Figure 14, there is shown the electropherograms for standard sample of
Batch produced SBE- -CD and SBE- -CD sample produced by continuous flow
synthesis. As can be seen, the continuous flow uses only 75% sodium hydroxide
compared to the amount used in the batch process (Stella et al, 1994), and a 7:1 molar
ratio of 1, 4-butane sultone to b-cyclodextrin instead of the 10:1 used by the prior art
method. The electropherogram for the continuous flow shows a positive shift of the
substitution envelope and change in the modal degree of substitution from ~ 6min to 8
min, and this indicates a higher average degree of substitution can be achieved more
economically.
Referring to Figure 15, there is shown there is shown the electropherograms for
standard sample of Batch produced SBE- -CD and SBE- -CD sample produced by
continuous flow synthesis. As can be seen, the continuous flow uses the same amount of
sodium hydroxide compared to the amount used in the batch process (Stella et al,
1994) nd a 7:1 molar ratio of 1, 4-butane sultone to b-cyclodextrin instead of the 10:1
used by the prior art method. The electropherogram for the continuous flow shows a
positive shift of the substitution envelope and a further change in the modal degree of
substitution from ~ 6min to 8.5 min, and this indicates a higher average degree of
substitution.
Referring to Figure 16, there is shown there is shown the electropherograms for
standard sample of Batch produced SBE- -CD and SBE- -CD sample produced by
continuous flow synthesis. As can be seen, the continuous flow uses 25% more sodium
hydroxide compared to the amount used in the batch process (Stella et al, 1994), and a
7:1 molar ratio of 1, 4-butane sultone to b-cyclodextrin instead of the 10:1 used by the
prior art method . The electropherogram for the continuous flow shows a positive shift
of the substitution envelope and further change in the modal degree of substitution
from ~ 6min to 8 min, very small population of lower degrees of substitution
(migration times ~2-7 min), and this indicates a higher degree of substitution.
Referring to Figure 17 there is shown the electropherograms for standard sample of
Batch produced SBE- -CD and SBE- -CD sample produced by continuous flow
synthesis. As can be seen, the continuous flow uses only 50% sodium hydroxide
compared to the amount used in the batch process (Stella et al, 1994), and an increase
from 7:1 to 10:1 molar ratio of 1, 4-butane sultone to b-cyclodextrin. As can be seen,
there are 10 peaks for the CF method and only 9 peaks for the batch method. The
number of peaks is indicative of the distribution of degree of substitution for the
derivatives. However, the electropherogram for the continuous flow shows an intense
peak at 5 minutes this possibly indicates the presence of a reaction impurity.
Referring to Figure 18, there is shown the electropherograms for standard sample of
Batch produced SBE- -CD and SBE- -CD sample produced by continuous flow
synthesis. The continuous flow uses only 75% sodium hydroxide compared to the
amount used in the batch process (Stella et al, 1994), and an increase from 7:1 to 10:1
molar ratio of 1, 4-butane sultone to b-cyclodextrin. The electropherogram for the
continuous flow shows a positive shift of the substitution envelope and change in the
modal degree of substitution from ~ 6min to 8 min, this indicates a higher average
degree of substitution.
Referring to Figure 19, there is shown there is shown the electropherograms for
standard sample of Batch produced SBE- -CD and SBE- -CD sample produced by
continuous flow synthesis. The continuous flow uses the same amount of sodium
hydroxide compared to the amount used in the batch process (Stella et al, 1994), and a
10:1 molar ratio of 1, 4-butane sultone to b-cyclodextrin, identical conditions used by
(Stella et al, 1994). The electropherogram for the continuous flow shows a positive shift
of the substitution envelope and a change in the modal degree of substitution from ~ 6
min to 8 min, a smaller population of lower degrees of substitution (migration time
range 2-7 minutes) and this indicates a higher degree of substitution. Comparing the
electropherogram at identical mole ratios of the material used, the flow method results
in species with higher degrees of substitution suggesting a more efficient and hence a
more economical production of cyclodextrin.
Referring to Figure 20, there is shown an electropherogram of batch-produced (Shah et
al, 2000) and continuous flow-produced standard SBE- -CD. The continuous process
used to produce the material shown in Figure 20 used 25% more sodium hydroxide
than the batch process (Stella et al, 1994), with an increase in the molar ratio of 1,4-
butane sultone to b-cyclodextrin from 7:1 to 10:1. Hence, the material produced by flow
synthesis is novel and demonstrates a positive skew in the Substitution Envelope with a
smaller population of lower degrees of substitution (migration time range 2-7 minutes)
and the modal degree of substitution changing from ~6 minutes to ~8 minutes. It is
concluded therefore that the continuous flow method of the invention results in an
increase in efficiency (more efficient activation of b-cyclodextrin hydroxyl groups by
sodium hydroxide; less consumption of 1, 4-butane sultone) resulting in a higher
degree of substitution.
A number of experiments were carried out, in order to fully explore the effect of
changing the b-CD: sodium hydroxide mass ratio, by altering the sodium hydroxide
content between 0%to 200% compared to the amount used in the batch process (Stella
et al, 1994). The results of this investigation have been highlighted in Table 3.
Table -The effect of changing the NaOH: b -CD mole ratio by the changing the NaOH
content; 100% nominal sodium hydroxide is equivalent to the base content used in US
5.376.645 (Stella etal I 4) .
Percentage NaOH: b -CD mole Observation(when reacted with butane sultone)
of NaOH ratio
%
0 - Very turbid unstable solution with solid white b -
CD precipitating out of solution
20 2:1 Less turbid immiscible solution with two layers
formed.
25 3:1 Less turbid immiscible solution with two layers
formed.
40 5:1 Less turbid immiscible solution with tiny butane
sultone particles suspended
50 6:1 Butane sultone reacts with the b -CD solution
forming a single phase homogenous solution
75 9:1 Butane sultone reacts with the b -CD solution
forming a single phase homogenous solution
100 11:1 Butane sultone reacts with the b -CD solution
forming a single phase homogenous solution
125 14:1 Butane sultone reacts with the b -CD solution
forming a single phase homogenous solution
150 17:1 Butane sultone reacts with the b -CD solution
forming a single phase homogenous solution,
solution becoming more viscous
160 l8:l Butane sultone reacts with the b -CD solution
forming a single phase homogenous solution,
solution becoming more viscous
175 20:l Butane sultone reacts with the b -CD solution
forming a single phase homogenous solution,
solution becoming more viscous
200 22:1 Butane sultone reacts with the b -CD solution
forming a thick viscous paste
In the absence of base (i.e. 0%NaOH), b-CD was insoluble and therefore did not react
with BS thus precipitating out. At 20-40% NaOH, the two phases would not mix, and b-
CD could not react fully with BS. At 150% NaOH, the dialysed product could not be
freeze-dried, and also the dialysis membrane was damaged by unreacted butane
sultone and the very basic condition arising from high concentrations of sodium
hydroxide, hence causing weakening and damaging the membrane. At 200% sodium
hydroxide, a viscous paste was formed that prevented pumping of the reaction
products. Hence, within the geometry of the apparatus used, 50-125% compared to the
amount used in the batch process (Stella et al, 1994), would allow SBE^-CD to be
manufactured using flow chemistry.
Example 2
The first application of SB^-CD in an injectable pharmaceutical drug product (i.e.
voriconazole) is described in the 2003 Pfizer patent, US 6,632,80361. The formulation
of an injectable form of voriconazole is described in Table 4.
Table 4 : Formulation of an injectable form of voriconazole using the 8BE-b-
platform
The manufacturing process is as follows:
1. Add SBE- -CD to 80% of the final volume of Water for Injections with constant
stirring until dissolved;
2. Add the voriconazole and stir until dissolved;
3. Make up the solution to its final volume (hence concentration) with the
remaining Water for Injections;
4. Filter the resulting solution through a sterilizing filter (o .22mpi pore size) into a
sterile container in a suitably validated GMP manufacturing area;
5. Fill into 20ml injection vials and stopper; and
6. Freeze-dry the product, stopper, over-cap and label.
The goal of this work was to try and develop innovative approaches to the manufacture
and application of SBE- -CD in the pharmaceutical industry. As can be seen from
Figure 23, use of the method of the invention means that three steps shown in Figure
22 can be omitted. It is evident on inspection of Figure 23 that by combining the two
processes, three commercial advantages arise:
1. It becomes unnecessary to transport SBE- -CD from the fine chemical
manufacturer to the customer. This would also include warehousing, etc.
2. SBECD could be manufactured on a just-in-time, just-enough basis.
3. One of the two expensive and time-consuming freeze-drying or spray drying
process steps could be avoided.
Example 3
The inventors have obtained Vapourtec flow chemistry equipment as illustrated in
Figure 21, which is more suited to commercial manufacture than the 'hand built'
reactors used to date. It was necessary to optimise the reaction to meet the
specification criteria set out in Shah's patent with respect to residual b-cyclodextrin and
1, 4-butane sultone. In addition, the SBE- -CD effluent stream was conditioned to
meet the requirements of pharmacopoeial Water for Injections monographs.
Example 4
As aqueous solutions of b -CD are intrinsically pyrogenic, the batch process requires
depyrogenation as part of the downstream purification. Using this CF manufacturing
method, it is possible to depyrogenate the system in reservoir (4) prior to reaction than
post reaction.
Summary
The results described herein demonstrated that the continuous flow process chemistry
is a more efficient way of producing SBECD and this is reflected in:-
(i) the average degree of substitution,
(ii) the low frequency of low degrees of substituted SBECD species;
(iii) production of SBECD with reduced quantities of starting materials;
(iv) the production of material, free from significant impurities, allowing
avoidance of quenching and intensive downstream processing.
The frequency of low degree of substituted SBECD species using the prior art batch
reaction is much higher than with using the continuous flow chemistry of the
invention. The continuous flow method of the invention enables a greater reaction
efficiency. The novel species produced by the continuous flow process have a higher
degree of substitution with a tighter distribution of substitution, as well as a higher
average degree of substitution per se.
Example
The CSTR-based Manufacturing Process
The aim of this work was to develop a continuous manufacturing process for the
manufacture of sulphobutylether b-cyclodextrin. It is known that mixing the b-
cyclodextrin and sodium hydroxide in a controlled way is important to the success of
the method of the invention. Firstly, it is important that both the aqueous, basified (i.e.
activated) b-cyclodextrin solution (4) is heated within the range of 6o-90°C prior to
mixing. Secondly, as b-cyclodextrin is added to the sodium hydroxide solution, a three
stage 'activation' process occurs: -
1) Firstly, it takes a finite time to add the b-cyclodextrin into the reservoir vessel
containing aqueous sodium hydroxide.
2) Next, the b-cyclodextrin dissolves in the sodium hydroxide solution.
3) Finally, and more significantly, an initial solution straw colouration
progressively 'deepens' which is considered to be a sign of completion of the
activation of the b-cyclodextrin by sodium hydroxide. With the deep
colouration present, and the reagents at the specified temperature, mixing then
proceeds.
The reaction proceeds in a continuous manner, i.e. once the pumps (8, 10) have started
they are not switched off until completion of the reaction. It is now generally
considered that the main reaction takes place in the first CSTR chamber (14). The
reaction takes place at a low temperature (65-ioo°C) and atmospheric pressure. The
CSTR-process handles the b-cyclodextrin-sodium hydroxide solutions and butane
sultone as an immiscible, two phase system.
It is known that some prior art methods create the conditions where butane sultone
and the aqueous b-cyclodextrin-sodium hydroxide streams may become miscible;
miscibility is generally considered to be an important process condition of flow
chemistry processing. Judging by the Average Degrees of Substitution achieved by
prior art methods, the goal of miscibility appears to have been achieved at the expense
of butane sultone stability which has led to very low Average Degrees of Substitution.
The method of the invention however involves carefully reacting sodium hydroxide
with b-cyclodextrin to activate it in advance of a two-phase continuous flow reaction,
and this is important in creating a highly efficient reaction and a controllable Average
Degree of Substitution in a small footprint. The activation process must be conducted
at elevated temperature (65-ioo°C) and for a specified time after the b-cyclodextrin has
dissolved in the aqueous sodium hydroxide solution. The activation process has
typically taken 30 minutes at this scale; the major indicator of completion is the colour
change which could be measured colourimetrically.
It is highly unlikely that this time and temperature dependent activation could be
achieved in any prior art batch or continuous flow methods. Whilst the reaction
procedure employs a CSTR, it is a surrogate for the use of a flow reactor with efficient
mixing and of suitable length to allow the reaction to complete within the reactor
tubing. The activation of b-cyclodextrin is an important process parameter prior to
reaction and this must continue irrespective of the reactor architecture.
Example 6
Analytical Methodology for High Degree of Substitution SBECD Species
The original work described herein was based on the capillary electrophoresis method
for sulphobutylether b-cyclodextrin described in the United States Pharmacopoeia
35/National Formulary 30 (USP35/NF30). The output of the analysis, the so-called
electropherogram, is shown in Figures 5 -20.
It can be seen that, whilst a qualitative idea of the substitution pattern is possible, it is
not easy to integrate the areas under the peaks reliably due to the shifting baseline. It is
also evident from Figure 14-16 and 18-20 that peak resolution deteriorates with
increasing substitution. Peaks appear to merge after approximately 8 minutes into the
run which makes it difficult to quantify the pattern of substitution. Furthermore, the
true nature of the substitution envelope could not be clearly understood.
Alternative methods have been proposed for the analysis of cyclodextrin derivatives
using high performance liquid chromatography (J. Szeman 2006). This has been
recently updated and applied to sulphobutylether b-cyclodextrin (J. Szeman 2012).
The method is based on a specialised ion-exchange HPLC column, CD-Screen-DAP,
where a bonded dimethylamino phenyl function includes in the eluting
sulphobutylether b-cyclodextrin to improve the selectivity of the analytical method.
High performance liquid chromatography with evaporative light scattering detection
(ELSD) is used for the separation of sulphobutylether b-cyclodextrin into its
substituted constituents in order to determine the average degree of substitution.
Identification of each substituted cyclodextrin is determined by comparing the
retention times of the standard, produced by the method described in US 6,153,746
(Shah, 2000), and tested according to the methods described in USP35/NF30 with that
of a material produced using the processing method described herein.
The chromatographic conditions are summarised as follows:
Reagents
1. Acetonitrile, HPLC grade
2. 0.5% triethylamine-acetic acid buffer, pH=5.o
Chromatographic Conditions
Instrument: Agilent 1100 series or equivalent HPLC instrument
Software: OpenLAB or similar system
Column: CD-Screen-DAP, 3mpi, 150 x 4.0mm, CDS-DAP-1504-03
Column temperature: 25°C ± i°C
Mobile phase A (MPA): 0.5% triethylamine-acetic acid buffer, pH=5
Mobile phase B (MPB): acetonitrile, HPLC grade
Flow rate: 1.0 ml/min
Gradient Ratio Time (min) 0 6 15
MPA(%) 100 50 50
MPB (%) 0 50 50
Detection: ELSD
Injection volume: 5 1
Concentration: 10 mg/ ml
Acquisition time: 15 minutes with post-time of 5 minutes
Needle wash: none
ELSD Conditions
Instrument: Alltech ELSD 2000 or equivalent ELSD instrument
Tube temperature: 115°C
Gas flow (nitrogen): 3.2 L/min
Gain: 2
Impactor: Off
Atypical chromatogram for the standard material produced using a prior art batch
method described in US 6,153,746 (Shah, 2000) is shown in Figure 24. Upon further
examination of Figure 24, it can be seen that material produced by the prior art
process has a range of substitution from Degree of Substitution 2 to 10. The Average
Degree of Substitution is 6.6.
The chromatogram for the sulphobutylether b-cyclodextrin produced using the method
of the invention and corresponding to Figure 20 is shown in Figure 25. It is readily seen
that a stable baseline is generated facilitating integration and subsequent processing of
the signal. Figure 25 indicates that material produced using the invention has a range
of substitution from Degree of Substitution 3 to 13. The Average Degree of Substitution
is 10.4, as described below.
In addition to producing sulphobutylether b-cyclodextrin with a higher Average Degree
of Substitution, the method of the invention, under these conditions, does not produce
any detectable di-substituted sulphobutylether b-cyclodextrin and produces significant
quantities of Degree of Substitution 11-13 not detected in the US 6,153,746 (Shah,
2000) material.
The inventors also have the corresponding HPLC traces corresponding to the
electropherograms in Figures 12 to 20. The power of the technique gives access to
descriptive statistics.
The Average Degree of Substitution is discussed herein using the standard method of
calculation. This method was modified for use with HPLC outputs and is explained
below.
The Individual Degree of Substitution (IDSn) is calculated using the following
formula:
IDSn = (PAn/ å PA) x 100 (1)
where å PA = åPAL+ PAL+1 ... PAH
n = Substitution Number
PA = Peak area
PAL = Peak area corresponding to lowest degree of substitution seen on the
chromatogram
PAH = Peak area corresponding to highest degree of substitution seen on
the chromatogram
These data can be used to describe an 'Envelope of Substitution' which is used as the
basis of a specification element in USP35/NF30, where each IDSn should fall within
the series of specified Proven Acceptable Ranges thus defining the 'Substitution
Envelope'.
The Individual Degree of Substitution metrics are then used to calculate the Average
Degree of Substitution as follows:
ADS = å (IDSn x n)/ 100
Table 1 shows data for the chromatogram shown in Figure 25. This can now be
processed using Equations 1-3 as follows:
Table 1: Integration table of the chromatogram of sulphobutylether b-cyclodextrin
produced by the method of the invention. Reaction conditions correspond to those
used to generate Figure 20 HPLC conditions are based on a gradient separation
with a CD-Screen-DAP column and ELSD detection
Individual Degree of Substitution - specimen calculation
åPA = åPAL + PAL+1 ... PAH
åPA = PA3 + PA4 + PA5 + PA6 + PA + As + PA + PA10 + PA + PA12 + A
åPA = 0.271 + O.507 + 1.455 + 3-142 + 5-221 + 13.283 + 24.842 + 46.056 +
53.920 + 39.220 + 16.570
åPA = 204.487
IDS3= (PA3/åPA) x l00 = (0.271/204.487) x 100 = 0.132527
IDS 4 = (RA4/SRA) x 100 = (0.507/204.487) x 100 = 0.247938
IDS 5 = (PA5/åPA) x 100 = (1.455/204.487) x 100 = 0.711537
IDS6 = (PA /åPA) x loo = (3.142/204.487) x 100 = 1.536528
IDS = (PA /åPA) x loo = (5.221/204.487) x 100 = 2.553219
IDSs = (PAs/åPA) x 100 = (13.283/204.487) x 100 = 6.495767
IDSg = (PA /åPA) x 100 = (24.842/204.487) x 100 = 12.14845
IDSio = (PA 0 / åPA) x loo = (46.056/204.487) x 100 = 22.5277
ID S = (PA / å PA) x loo = (53.920/204.487) x 100 = 26.36842
IDS 2 = (PA 2/ å PA) x 100 = (39.220/204.487) x 100 = 19.1797
IDS 3 = (PA 3/ å PA) x 100 = (16.570/204.487) x 100 = 8.103205
Average Degree of Substitution - specimen calculation
ADS = å(IDSn x substitution number)/ 100
n = substitution number
IDSn x substitution number
IDS 3X3 = 0.132527x 3 = 0.397580
IDS 4 x 4 = 0.247938 x 4 = 0.991750
IDS5 x 5 = 0.711537x 5 = 3·557683
IDS6 x 6 = 1.536528 x 6 = 9.219168
IDS x 7 = 2.553219 x 7 = 17.872530
IDSs x 8 = 6.495767 x 8 = 51.966140
IDSg x 9 = 12.14845 x 9 = 109.336046
IDS10 x 10 = 22.5277 x 10 = 225.227032
IDS 11X11 = 26.36842 x 11 290.052668
IDS12 x 12 = 19.1797 x 12 = 230.156440
IDS13 x 13 = 8.103205 x 13 105.341660
å(IDSn x substitution number) = (IDS3 x 3) + (IDS4 x 4) + (IDS5 x 5) + (IDS6
x 6) + (IDS x 7) + (IDSs x 8) + (IDS x 9) + (IDS 0 x 10) + ( ID S x 11) + (IDS 2 x
i2) + (IDS 3 x i3)
å(IDSn x substitution number) = 0.397580 + 0.991750 + 3.557683 +
9.219168 + 17.872530 + 51.966140 + 109.336046 + 225.227032
+ 290.052668 + 230.156440 + 105.341660
å(IDSn x substitution number) = 1044.118697
ADS = å(IDSn x substitution number)/ 100 = 1044.118697/ 100 =
10.44
Average Degree of Substitution = 10.4
The material described in Figure 25 therefore has an average degree of substitution of
10.4, which is substantially higher than material produced by batch manufacture or
fully continuous flow process.
Example 7
The Manipulation of Average Degree of Substitution Using Sodium Hydroxide
The samples of sulphobutylether b-cyclodextrin that have been produced have now
been reanalysed by HPLC. The data has been processed to generate the Average
Degrees of Substitution. The table shown in Figure 26 shows a summary of the data
adding the Average Degree of Substitution data and dispersion data.
In general, it can be seen from the Table in Figure 26 that an increase in the content of
sodium hydroxide will increase the Average Degree of Substitution of sulphobutylether
b-cyclodextrin. Furthermore, the more extreme CSTR reactions produce material with
Average Degree of Substitution at levels not previously seen using batch or continuous
flow reactions. The higher Average Degree of Substitution arises due to the presence of
highly substituted species with an Individual Degree of Substitution in excess of 10.
The table shown in Figure 27 describes an attempt to produce material compliant with
the USP35/NF30 monograph with the use of more moderate reaction conditions.
Analysis indicated that the IDSn 'envelope' present in the CSTR 10:1 butane sultone: b-
cyclodextrin molar ratio and a 6:1 Sodium hydroxide to b-cyclodextrin molar ratio
reaction produced the nearest match. It can be seen that, using the HPLC method, the
US 6,153,746 (Shah, 2000) material broadly complies with the specification. The
material produced by the CSTR method is less compliant due to a more symmetrical
distribution of IDSn. Whilst compliant material has yet to be produced, the ability to
control the process with respect to stoichiometry indicates that, with further process
refinement, this should be possible.
Example 8
Novelty of SBE- -CD prepared using the method of the invention
Historically, the number of pendant sulphobutyl groups on the cyclodextrin determines
the Individual Degree of Substitution metric and the Substitution Envelope. The
weighted average of the abundance of each species gives rise t o the Average Degree of
Substitution metric. There are three possibilities for defining the novelty of the SBE- b-
CD prepared using the method of the invention:
a) Substitution
What is really important, chemically, is the number of cyclodextrin rings available t o
form inclusion complexes with drugs, because this is what makes the cyclodextrin work.
Whilst the parent beta cyclodextrin gives the greatest number of rings for a given
molecular mass, it is believed t o b e nephrotoxic and this makes substitution necessary.
It is known that increasing the degree of substitution increases the aqueous solubility of
the cyclodextrin and high solubility of the cyclodextrin is a pre-requisite t o achieving a
high payload drug solubility. The following table summarises substitution molecular
mass ratios:
Beta Butane Substitution
cyclodextrin Proton Molecular sultons Molecular Molecular
IDS MWoleeicguhltar Loss WPreoigtohnt ALfotsesr ConGtrriobuuption WeiIgDhSt for FrMacatsison
1 1134.98 - 1 1133.98 136.17 1270.15 0.11
2 1134.98 -2 1132.98 272.34 1405.32 0.19
3 1134.98 -3 1131.98 408.51 1540.49 0.26
4 1134.98 -4 1130.98 544.68 1675.66 0.32
5 1134.98 -5 1129.98 680.85 1810.83 0.37
6 1134.98 -6 1128.98 817.02 1946.00 0.42
7 1134.98 -7 1127.98 953.19 2081.17 0.45
8 1134.98 -8 1126.98 1089.36 2216.34 0.49
9 1134.98 -9 1125.98 1225.53 2351.51 0.52
10 1134.98 -10 1124.98 1361.70 2486.68 0.54
11 1134.98 -11 1123.98 1497.87 2621.85 0.57
12 1134.98 -12 1122.98 1634.04 2757.02 0.59
13 1134.98 -13 1121.98 1770.21 2892.19 0.61
The molecular mass of beta cyclodextrin is 1134.98 Dalton. To create a monosubstituted
beta cyclodextrin, a proton is removed, and replaced with a linear butane
sultone function with a molecular mass of 136.17 Dalton. The resulting molecular mass
of individual degree of substitution (IDS), where n=i is 1270.15 Dalton. If one considers
5 the mass associated with the cyclodextrin ring as a fraction of the total mass, it is
possible to calculate a Substitution Molecular Mass Fraction. This means that 11% of
the mass is associated with the substituent functions (or 89% is associated with the
cyclodextrin ring function). The table shows these values up to the SBE- -CD prepared
using the method of the invention, having a surprisingly high IDS = 13 species.
10
The values of individual degree of substitution (IDS) and Substitution Molecular Mass
Fraction (SMF) shown in the above table have been plotted out on Figure 28, and the
relationship can be seen. The inventors believe that, to date, SBE- -CD with an SMF
greater than 0.57 has not been previously reported, and as such the composition is
15 novel per se.
b ) Molecular Weight
This is the first report of a derivatised species with a molecular weight in excess of
2486.68 Dalton or within the range 2621.85 - 2892.19. Referring to Figure 29, there is
0 shown the relationship between individual degree of substitution and molecular weight.
Molecular weight is believed to be an alias for Individual Degree of Substitution and so
the Substitution Molecular Mass Fraction (SMF) may be the better choice.
c) Substitution Envelope
25 When considering Column 2 of Figure 27, it is possible to define a novel peak area limit
as follows:
USP-NF USP-NF Novel Novel
Peak Area Peak Area Peak Area CSTR
IDSn Peak Area Percentage Range PercentagePercentage Range 10:1 and
Percentage Upper Lower Upper +25%
Lower Limit
Limit Limit Limit
1 ().()() 0.30 0.30 0.00 0.00 0.00 0.00 0.00
2 ().()() 0.90 0.90 0.13 0.00 0.00 0.00 0.00
3 0.50 5.00 4.50 0.88 0.00 0.30 0.30 0.10
4 2.00 10.00 .00 4.91 0.00 0.90 0.90 0.20
5 10.00 20.00 10.00 14.61 0.50 5.00 4.50 0.70
15.00 25.00 10.00 iiiiiiii 0.50 5.00 4.50 1.50
20.00 .¾().()() 10.00 29.50 0.50 5.00 4.50 2.60
10.00 25.00 15.00 1 . 1 2.00 10.00 8.00 6.50
2.00 12.00 10.00 4.99 10.00 20.00 10.00 12.10
0.00 4.00 4.00 0.51 15.00 25.00 10.00 22.50
0.00 0.00 0.00 0.00 20.00 30.00 10.00 26.40
0.00 0.00 0.00 0.00 10.00 25.00 15.00 19.20
0.00 0.00 0.00 0.00 2.00 12.00 10.00 8.10
0.00 0.00 0.00 0.00 0.00 4.00 4.00 0.00
The USP-NF Peak Area Percentage describes a series of Proven Acceptable Ranges for
an upper and lower distribution of IDSn in which a 'Substitution Envelope' resides.
With a shift in IDSn to higher values using the process of the invention, it is possible to
shift the envelope. As shown in Figure 27, it is possible to 'de-tune' the process of the
invention to broadly comply with the USP-NF Envelope, and this is not possible using
the fully batch or fully continuous processes described in the prior art.
Summary
Using a novel, improved HPLC analytical method, the inventors have validated their
earlier observations described herein. The technique has allowed them to produce
descriptive statistics for high degree of substitution material. The sulphobutylether b-
cyclodextrin composition, produced by the CSTR process according to the invention
described herein, is novel in two respects: (i) it has an unprecedented high average
degree of substitution; and (ii) the existence of highly substituted species with IDSn
higher than 10. The CSTR process depends upon pre-activation of the b-cyclodextrin
feedstock by sodium hydroxide where the extent of activation determines the Average
Degree of Substitution. The process allows control of Average Degree of Substitution
by varying the sodium hydroxide concentration. The process can be used to produce
material with a high Average Degree of Substitution. It will be possible to manufacture
material compliant with the USP35/NF30 specification for sulphobutylether b-
cyclodextrin. The process enables the production of sulphobutylether b-cyclodextrin on
a 'just in time', 'just enough' basis in a small manufacturing footprint.
REFERENCES
J . Szeman, K. Csabai, K. Kekesi, L. Szente, G. Varga. "Novel stationary phases for highperformance
liquid chromatography." Journal of ChromatographyA, 2006:
76-82.
an, T. Sohajda, E. Olah, E. Varga, K. Csabai, G. Varga, L. Szente.
"Characterization of Randomly Substituted Anionic Cyclodextrin Derivatives
with Different Analytical Methods." 16th International Cyclodextrin
Symposium. Tianjin, China, 2012.

Claims
1. A method for preparing sulphoalkyl ether- b-cyclodextrin, the method
comprising contacting cyclodextrin with a base to form activated cyclodextrin, and
separately contacting the activated cyclodextrin with an alkyl sultone to form
sulphoalkyl ether- b-cyclodextrin, characterised in that the sulphoalkylation reaction is
carried out under continuous flow conditions.
2. A method according to claim 1, wherein the activation reaction is carried out as
a batch process.
3. A method according to either claim 1or claim 2, wherein the base comprises an
alkali metal hydroxide, such as sodium hydroxide, lithium hydroxide or potassium
hydroxide, preferably sodium hydroxide.
4. A method according to any preceding claim, wherein the molar ratio of base to
cyclodextrin is within the range of 2:1to 22:1, or 6:1to 20:1, or 6:1 to 15: 1.
5. A method according to any preceding claim, wherein the method comprises
controlling the average degree of substitution (ADS) of sulphoalkyl ether b-cyclodextrin
in the sulphoalkylation reaction by varying the base concentration in the activation
reaction.
6. A method according to any preceding claim, wherein the cyclodextrin is a-, b-
or g -cyclodextrin.
7. A method according to any preceding claim, wherein cyclodextrin is b-
cyclodextrin.
8. A method according to any preceding claim, wherein the alkyl sultone
comprises 1, 4-butane sultone.
9. A method according to any preceding claim, wherein the sulphoalkyl ether- b-
cyclodextrin comprises sulphobutyl ether b-cyclodextrin (SBE^-CD).
10. A method according to any preceding claim, wherein the activation reaction is
conducted at atmospheric pressure.
11. A method according to any preceding claim, wherein the activation reaction is
carried out in a first reservoir vessel at a temperature of about 50 to 95 °C, preferably
60 to 70°C.
12. A method according to claim 11, wherein the alkyl sultone is contained within a
second reservoir vessel, and the first and second vessels are not directly connected to
each other, such that the sultone and the base do not react with each other.
13. A method according to claim 12, wherein the activated cyclodextrin and the
alkyl sultone are fed to a confluent 3-way junction where they are allowed to react to
produce the substituted sulphoalkyl ether b-cyclodextrin.
14. A method according to any preceding claim, wherein the molar ratio of sultone
to cyclodextrin is between about 7:1 and 33:1, preferably 7:1 to 17:1.
15. A method according to any preceding claim, wherein the sulphoalkylation
reaction is conducted at a temperature of 60 to 100 °C, preferably 60 to 70°C.
16. A method according to any preceding claim, wherein the sulphoalkylation
reaction is conducted at atmospheric pressure.
17. A method according to any preceding claim, wherein the alkylation reaction is
carried out in a continuous stirred tank reactor (CSTR).
18. A method according to any preceding claim, wherein the average degree of
substitution (ADS) of the sulphoalkyl ether b-cyclodextrin that is produced is greater
than 7, preferably 7.3 or more, more preferably 8 or more, even more preferably 9 or
more, and most preferably 10 or more.
19. Use of sodium hydroxide concentration for controlling the average degree of
substitution (ADS) of sulphoalkyl ether b-cyclodextrin produced in a sulphoalkylation
reaction between activated cyclodextrin and an alkyl sultone.
20. Sulphoalkyl ether- b-cyclodextrin obtained or obtainable by the method
according to any one of claim 1-18.
21. A composition comprising sulphobutyl ether b-cyclodextrin (SBE-P-CD),
wherein the average degree of substitution (ADS) is 7.3 or more, preferably 8 or more,
even more preferably 9 or more, and most preferably 10 or more.
22. A composition according to claim 21, wherein the sulphobutyl ether b-
cyclodextrin (SBE-P-CD) is produced by the method according to any one of claim 1-18.
23. A composition according to either claim 21 or claim 22, wherein the
composition comprises SBE-P-CD having a Substitution Molecular Mass Fraction
(SMF) greater than 0.57, more preferably greater than 0.58, and even more preferably
greater than 0.59.
24. Use of the sulphoalkyl ether b-cyclodextrin according to claim 20, or the
composition according to any one of claims 21-23, a drug delivery system.
25. Use according to claim 24, wherein the drug delivery system is an excipient,
which exhibits little or no side effects with regard to renal physiology.
26. Use according to either claim 24 or claim 25, wherein the sulphoalkyl ether- b-
cyclodextrin comprises sulphobutyl ether b-cyclodextrin (SBE-P-CD).
27. A pharmaceutical excipient comprising the sulphoalkyl ether b-cyclodextrin
according to claim 20, or the composition according to any one of claims 21-23.
28. An excipient according to claim 27, wherein the sulphoalkyl ether- b-
cyclodextrin comprises sulphobutyl ether b-cyclodextrin (SBE^-CD).
29. A method of preparing a pharmaceutical composition, the method comprising
preparing the pharmaceutical excipient according to either claim 27 or claim 28, and
contacting the excipient with an active pharmaceutical ingredient (API) to produce a
pharmaceutical composition.
30. A method according to claim 29, wherein the active pharmaceutical ingredient
comprises voriconazole, ziprasidone, aripiprazole, maropitant, amiodarone, or
carfrlzomib, or their salts, solvates, polymorphs, pseudopolymorphs or co-crystals.