# An NMR Study of the Bortezomib Degradation under Clinical Use Conditions

Authors: Adele Bolognese; Anna Esposito; Michele Manfra; Lucio Catalano; Fara Petruzziello; Maria Carmen Martorelli; Raffaella Pagliuca; Vittoria Mazzarelli; Maria Ottiero; Melania Scalfaro; Bruno Rotoli
Journal: Advances in Hematology
 (2009)

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## Abstract

The (R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl-boronic acid, bortezomib (BTZ), which binds the 20S proteasome subunit and causes a large inhibition of its activity, is a peptidomimetic boronic drug mainly used for the treatment of multiple myeloma. Commercial BTZ, stabilized as mannitol derivative, has been investigated under the common conditions of the clinical use because it is suspected to be easily degradable in the region of its boronic moiety. Commercial BTZ samples, reconstituted according to the reported commercial instructions and stored at 4∘C, were analyzed by high-field nuclear magnetic resonance spectroscopy in comparison with identical samples bubbled with air and argon, respectively. All the samples remained unchanged for a week. After a month, the air filled samples showed the presence of two main degradation products (6% of starting material), the N-(1-(1-hydroxy-3-methylbutylamino)-1-oxo-3-phenylpropan-2-yl) pyrazine-2-carboxamide (BTZ1; 5%, determined from NMR integration) and the (S)-N-(1-(3-methylbutanamido)-1-oxo-3-phenylpropan-2-yl)pyrazine-2-carboxamide (BTZ2; 1%, determined from NMR integration), identified on the basis of their chemical and spectroscopic properties. The BTZ1 and BTZ2 finding suggests that, under the common condition of use and at 4∘C, commercial BTZ-mannitol is stable for a week, and that, in time, it undergoes slow oxidative deboronation which partially inactivates the product. Low temperature and scarce contact with air decrease the degradation process.


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## Body

## 1. Introduction

Bortezomib (BTZ) [1], the (R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)
propanamido)butylboronic acid, (BTZ,
Figure 1), is one of the most important members of a new class of drugs,
containing a boronic acid moiety, effective on a wide group of tumors. At
present, it is mainly used for the treatment of multiple myeloma, a plasma cell
tumor which accounts for 10% of all blood system malignancies [2, 3]. BTZ is a peptidomimetic compound,
constituted by a modified leucine-phenylalaninedipeptide, containing a boronic
acid at the C-terminal. It is able to interact with proteasome, an
intracellular apparatus which brakes down damaged or unneeded proteins,
inhibiting the proteolysis action [4, 5].Figure 1
(R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2- carboxamido)-propanamido)butylboronic acid.As a boronic acid [6], BTZ shows high affinity for hard oxygen-containing
nucleophiles according to the Lewis hard-soft acid-base theory. Specifically,
peptide boronates are well-known inhibitors of serine proteases, forming a
serine-boronate tetrahedral transition state complex. BTZ, active at subnanomolar concentrations (Ki 0.6 nM), seems to
interact with the hydroxyl of a threonine present in the active site of the
N-terminal of the 20S β5 subunit of the proteasome. Formation of a
tetrahedral complex (X) inhibits the
chymotryptic proteolytic activity, totally hampering proteasomal functions
(Figure 2).Figure 2
Formation of tetrahedric complex
between the hydroxyl group of threonine present on the 20Sβ5 subunit of the
proteasome and the boronic residue of BTZ.
A water molecule is involved in the mechanism of complex formation [6–8].Generally, boronic acids are
compounds characterized by a vacant 2p orbital. This electron deficiency
determines a chemical instability resulting in the formation of tetrahedral
boron adducts owing to the attack of nucleophile agents, such as water,
hydroxide, alkoxide, or amines. At room temperature, stable cyclic esters with
saccharides are also formed through rapid and reversible reactions. Moreover,
the aminoalkylboronic acids, boron analogues of common amino acids, asBTZ is, undergo a spontaneous
1,3-rearrangement to give the homologated amines, owing to the instability of
free α-amino groups possessing hydrogen substituents.
These compounds yield boric acids and alcohols by degradation and undergo
oxidative reactions which easily destroy the C–B bond longer and
weaker than the corresponding C–C bond. Figure 3 illustrates this
characteristic reactivity (reactions a and b), which is of some interest for
BTZ chemical stability [9–11].Two possible degradation processes ofBTZ in its pharmaceutical solution form (a) degradation pathway of
boronic amino acids in presence of water or other nucleophilic agents; (b)
oxidative degradation pathway of boronic acids.
(a)(b)The aim of this study is to
explore the chemical stability of a commercialBTZ sample (Velcade) in its pharmaceutical form (i.e., stocked as a
sterile, lyophilized formulation with mannitol as a bulking agent, in a glass
vial filled with nitrogen), after its reconstitution according to the
commercial reported instructions and stored at 4°C.


## 2. Materials and Methods

BTZ is commercialized by
Millennium Pharmaceuticals (Mass, USA) in the US and Janssen-Cilag in Europe under the trade
name Velcade, and is administered as intravenous bolus. The vials are
reconstituted with 3.5 mL of sterile NaCl 0.9% to produce 1 mg/mL of BTZ and 10 mg/mL of mannitol. The product information states that reconstituted BTZ is
stable for 8 hours when stored at<25C° and protected from light, and for 3 hours
in a syringe.The samples ofBTZ under investigation were
reconstituted using a sterile NaCl 0.9% solution (in deuterated water D2O,
Merck) to produce the suitable NMR samples, according to the commercial
reported instructions.Sample A was used for a direct NMR investigation without further
treatment, while the tubes containing the samples B and C were filled with
argon (B) and oxygen (C), respectively. All the experiments
were performed in triplicate.The solutions A, B, and C, kept in dark at4°C, were subjected to H1
NMR investigation and tested during a week to record possible differences in
the mixture composition. After one month, the previously examined samples A, B, and C, kept in dark at 4°C, were reanalyzed by H1 NMR spectroscopy.


### 2.1. NMR and HPLC Experiments

Nuclear magnetic resonance
(NMR) spectra were recorded at 500 MHz for  [1H]  and 12 MHz forC13 on a Fourier
Transform NMR Varian 500 Unity Inova spectrometer. Carbon multiplicity was
evidenced by DEPT experiments. HPLC analysis was performed at room temperature
(~25C°) using a Shimadzu LC-6A pump equipped with Rheodyne 7215 injection
valve 20-mL, and a Shimadzu SPD-6A spectrophotometric detector working at 280 nm; a Symmetry C18 Waters column was employed. The mobile phase consisted of
40% (v/v) acetonitrile and 60% (v/v) 30 mM KH2PO4H3PO4 (pH 3.0). The product was eluted
at 3.9 minutes with a flow rate of 1 mL/min.


## 2.1. NMR and HPLC Experiments

Nuclear magnetic resonance
(NMR) spectra were recorded at 500 MHz for  [1H]  and 12 MHz forC13 on a Fourier
Transform NMR Varian 500 Unity Inova spectrometer. Carbon multiplicity was
evidenced by DEPT experiments. HPLC analysis was performed at room temperature
(~25C°) using a Shimadzu LC-6A pump equipped with Rheodyne 7215 injection
valve 20-mL, and a Shimadzu SPD-6A spectrophotometric detector working at 280 nm; a Symmetry C18 Waters column was employed. The mobile phase consisted of
40% (v/v) acetonitrile and 60% (v/v) 30 mM KH2PO4H3PO4 (pH 3.0). The product was eluted
at 3.9 minutes with a flow rate of 1 mL/min.


## 3. Results and Discussion

Solutions A, B, and C, kept in the dark at4°C, tested at intervals for
a week by HPLC, did not differ between the initial and the final stage (Figure 6(a)). After one month, a small
amount of two products eluted at 1 and 5 minutes was recorded (Figure 6(b)). According to this evidence, no
change was observed in the NMR samples under the same conditions. After one
month at 4°C,
NMR reanalysis of the same samples A, B, and C showed that A and B were
unchanged, whereas some modifications had taken place in the spectrum of
solution C.Particularly, the proton (H̲,
Figure 1) multiplet signal at 3.31δ attributed to the hydrogen on the carbon linked
to boron decreased in intensity by about 5%.To investigate whether the
origin of this difference was due to the presence of oxidation decomposition
products, an air flow was gently bubbled, for six hours, through aBTZ sample reconstituted according to
the commercial instructions and kept at 4°C.The mixture was extracted
with chloroform and analyzed chromatographically by HPLC. Together with the
mainBTZ, two new products, BTZ1 and BTZ2 (resp., ~5% and 1% of starting BTZ), were recovered and investigated by NMR spectroscopy (see Figures 4 and 5). BTZ1 showed an H1 NMR spectrum (CDCl3) with signals at δ9.38 (1H,
d; J  =  1.5 Hz), 8.73 (1H, d; J  =  2.6 Hz), 8.48 (1H, m), 8.28 (1H, bs), 8.02 (1H,
bs), 7.26 (5H, m), 5.36 (1H, dd; J  =  7, 5.5 Hz), 4.70 (1H, dd; J  =  5.1, 9.0 Hz), 3.20
(1H, dd; J  =  14, 5.1), 3.02 (1H, dd; J  =  14, 9.1), 1.71 (1H, dd; J  =  7, 14.4),
1.66 (1H, dd; J  =  5.5, 14.4), 1.52 (1H, m), and 0.98 (6H, d). The C13 NMR spectrum
showed signals at δ171.3, 164.7, 148.5, 145.6, 144.7, 137.6, 135.9,
130.7 (2C),
129.1, 127.5 (2C),
74.4, 54.2, 43.6, 38, 24.1, and 22.4 (2C).
The Mass spectra of BTZ1 obtained
from MALDI spectra recorded a peak at 339 m/e corresponding to the more stable
protonated-dehydrated ion, (M+H−H2O, 100 %).Figure 4
N-(1-(1-hydroxy-3-methylbutylamino)-1-oxo-3- phenylpropan-2-yl) pyrazine-2-carboxamide (BTZ1).Figure 5
(S)-N-(1-(3-methylbutanamido)-1-oxo-3- phenylpropan-2-yl)pyrazine-2-carboxamide
(BTZ2).HPLC analysis of bortezomib
solutions A, B, and C kept in dark at4°C for a week. (a) No degradation product was observed. (b) After a month, a
small amount of two new products, eluted at 1 and 5 minutes, respectively, was
recorded.
(a)(b)On the basis of the chemical
and spectroscopic properties [12–16], the product BTZ1 was identified as the N-(1-(1-hydroxy-3-methylbutylamino)-1-oxo-3-phenylpropan-2-yl)
pyrazine-2-carboxamide. The decreasing intensity (about 5% in a month) of the
signal at 3.31δpresent in the unchanged BTZ (H̲ in Figure 1) recorded into the H1 NMR
spectrum represents a clear indication that a chemical changing had happened at
the carbon holding the hydrogen corresponding to this signal.Moreover, the reproducibility
of theH1 NMR spectra of samples A and B in time, clearly
in contrast with the degradation of sample C, suggests that the oxygen bubbled into the C sample tube
plays a determining role in the BTZ stability. According to the chemical behavior of boronic acid reported in
literature, BTZ is sensitive to the
oxidative effect of oxygen present in air and undergoes oxidative deboronation
to BTZ1.Small amount (1% of startingBTZ) of another degradation product BTZ2, detected in the sample C, was recovered and investigated by NMR
spectroscopy. BTZ2 showed an H1
NMR spectrum (CDCl3) with signals at δ9.38 (1H,
d; J  =  1.5 Hz), 8.73 (1H, d; J  =  2.6 Hz), 8.67 (1H, bs), 8.48 (1H, m), 8.02 (1H,
bs), 7.28 (5H, m), 4.91̲ (1H̲, dd; J = 5.1, 9.1 Hz), 3.18 (1H, dd;
J  =  14.0, 5.1), 3.12 (1H, dd; J  =  14.0, 9.1), 2.09 (2H, m), 1.72 (1H, m), and 1.02 (6H,
d). The C13 NMR spectrum showed signals at δ172.1, 171.3,
164.7, 148.5, 145.6, 144.7, 137.6, 135.9, 131.7 (2C), 129.3, 127.8 (2C), 55.1, 46.3, 38, 25.1, and 22.5
(2C).The Mass spectra ofBTZ2 obtained from ES and MALDI spectra
recorded a peak at 335 m/e corresponding to the protonated (M+H+100%). Main
characteristics of BTZ, BTZ1, and BTZ2 are summarized in Table 1.Table 1
Molecular
weight, mass peak, and elemental analysis ofBTZ, BTZ1, and BTZ2.
Comp.Elemental analysisMWMass peak m/zCHNcalcdfoundcalcdfoundcalcdfoundBTZC19H25BN4O4[M+H−H2O]+59.3959.486.566.5814.5814.61384367BTZ1C19H24N4O3[M+H−H2O]+64.0364.126.796.8115.7215.70356339BTZ2C19H22N4O3[M+H]+64.3964.326.266.3015.8115.78354355On the basis of the reported data, the structure of N-(1-(3-methylbutanamido)-1-oxo-3-phenylpropan-2-yl)pyrazine-2-carboxamide
was attributed toBTZ2 [12–16].Our results suggest that even
after reconstitution with saline, BTZ solutions are stable for at least one
week in the dark at4°C,
and that very small amounts of BTZ derivatives are detectable in vials stored
in the presence of oxygen after one month only. Thus, residual amounts of the drug
in vials used for therapeutic purposes can be stored and reutilized within a
few weeks, on the same patient or on different ones, without detectable loss of
potency. These results may have interesting implications, both for patient
management and in terms of cost effectiveness, particularly for centers
treating small numbers of patients, considering the high cost of BTZ therapy.


## 4. Conclusions

The BTZ compound present in a reconstituted sample of Velcade kept at4°C in dark resulted stable for a week, whereas it
underwent oxidative transformation in presence of high air (oxygen)
concentration after a month. The present result is not unexpected given the widely
reported lability of boronic acids toward the oxidative deboronation.



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*Source: 704928-2009-04-14.xml*