Keywords imipenem - cilastatin sodium - stability - oxidation - free water
Introduction
Imipenem and cilastatin sodium for injection (IMI/CIL), as the first carbapenem drug
used in clinic, is used to treat complicated intra-abdominal infections and complicated
urinary tract infections including pneumonia and other serious bacterial infections
in adults. Compared with other types of antibacterial drugs, imipenem has higher sensitivity
to pathogenic bacteria while bringing better safety. Therefore, it has important clinical
value.[1 ] However, as a β-lactam drug, the chemical stability of IMI/CIL is very unstable,
hence it is necessary to pay more attention to the factors affecting its stability.
Oxidation is a common pathway for drug degradation.[2 ] Active pharmaceutical ingredients (APIs) exposed to the air tend to deteriorate
due to the influence of oxygen,[3 ] thus decreasing drug potency and greatly reducing their shelf life. In addition
to oxygen, water is another important factor that influences the chemical stability
of solid pharmaceuticals. Usually the degradation rate of a drug is dependent on the
water content.[4 ] Although some studies have assessed the effects of oxygen and water on the degradation
of compounds,[3 ]
[5 ] however, the influence of oxygen and water on the stability of IMI/CIL has not been
evaluated.
In previous studies, it was generally believed that total water was the main factor
affecting drug degradation, and few studies have studied the effect of free water
on drug degradation. In this study, the effects of headspace oxygen (HO) level and
water content on IMI/CIL were investigated. Moisture-mediated interactions between
cilastatin and imipenem were also explored. This study demonstrated that free water
content is a better predictor of the safety and stability of imipenem and cilastatin
sodium than total water content. Moreover, the degradation mechanism of imipenem and
cilastatin sodium exposed to oxygen and moisture has been proposed based on molecular
structure information. The effects of shape and size of drug particles on the stability
of imipenem and cilastatin sodium were also investigated. Our data suggested that
the greater the surface area of imipenem and cilastatin sodium exposed in the air,
the greater the effect of sodium cilastatin on imipenem. This work would contribute
to the production of IMI/CIL with improved stability.
Materials and Methods
Materials
Imipenem, cilastatin sodium, and sodium bicarbonate were provided by manufacturer
A. IMI and CIL from three batches (2021–1, 2021–2, 2021–3) were prepared in different
processes. Acetonitrile was purchased from Honeywell International (New Jersey, United
States) and phosphoric acid (H3 PO4 ) was from Sinopharm Group Co. Ltd (Shanghai, China). Anhydrous sodium dihydrogen
phosphate and anhydrous sodium dihydrogen phosphate were obtained from General Reagent.
All laboratory water was prepared by using a Millipore milli-Q water purification
system (Merk KGaA, Darmstadt, Germany). All other chemicals were of reagent grade
and used without further purification.
Analysis of Imipenem and Cilastatin Sodium Impurities
A high-performance liquid chromatography (HPLC) method was used for analyzing the
contents of imipenem, cilastatin sodium, and their related substances. Analyses were
performed on a Thermo DIONEX Ultimate 3000 HPLC system (Thermo Fisher Scientific,
Bremen, Germany) equipped with an LPG-3400SDN Ternary Pump, a TCC-3000RS column compartment,
a WPS-3000TSL ANALYTICAL autosampler, and a diode array detector (all the above instruments
were from Thermo Fisher Scientific). Chromatography was conducted on a C18 column
(250 mm × 4.6 mm, 5 µm, Waters, United States). Eluent A was composed of potassium
dihydrogen phosphate–acetonitrile (99.3:0.7, v/v) with pH = 7.3 adjusted by H3 PO4 , and eluent B was potassium dihydrogen phosphate–acetonitrile (50:50, v/v) with pH = 7.3.
A gradient elution was performed using the method described in [Table 1 ]. The injection volume was 10 μL and the column temperature was maintained at 30°C.
The flow rate was set to 1.0 mL/min. The UV detection was performed at 260 nm. Parameters
such as retention time, resolution, and symmetry factor verified the suitability of
the system. All the sample solutions must be prepared immediately before analysis.
Table 1
Gradient elution method
Time (min)
Mobile phase A%
Mobile phase B%
0
100
0
3
100
0
28
90
10
38
90
10
63
50
50
78
30
70
88
30
70
89
100
0
97
100
0
IMI/CIL Preparation
IMI/CIL was prepared according to a reported study, as well as the prescription including
500 mg of imipenem, 500 mg of cilastatin sodium, and 20 mg of sodium bicarbonate.[6 ]
Effect of HO Levels on Stability
Preparation of IMI/CIL with Different HO Levels
To prepare IMI/CIL with different HO levels, vials filled with imipenem and cilastatin
from 2021–1 were purged with air, premixed oxygen/nitrogen gas (5%/95%), or high-purity
nitrogen (99.999%) at 20, 5, or 2% oxygen levels, respectively, and then sealed and
capped promptly. These samples were stored at 60°C and analyzed for the major degradation
substances by HPLC on day 0, 10, and 30.
Preparation of IMI/CIL from Different Batches at the Same HO Level
IMI/CIL samples with the same HO level of 2% from 2021–1, 2021–2, and 2021–3 were
prepared by purging with nitrogen. The samples were kept at 60°C and analyzed for
the major degradation substances by HPLC on day 0, 5, 10, and 30.
Effect of Water Content on Stability
To explore the effect of water on the stability of imipenem and cilastatin sodium,
imipenem, cilastatin sodium, and IMI/CIL were placed in different humidity environments.
Water contents were measured by thermogravimetric analysis (TGA) and loss on drying
(LOD) method. The major degradation substances of imipenem-cilastatin sodium were
detected by HPLC, and the effect of water on the stability of imipenem-cilastatin
sodium was studied. Imipenem, cilastatin sodium, and IMI/CIL were dried to investigate
the effect of reducing their free water content on the stability of IMI/CIL.
Treatment of Imipenem, Cilastatin, and IMI/CIL
To evaluate the effect of moisture on the stability of imipenem and cilastatin sodium,
imipenem, cilastatin sodium, and IMI/CIL were stored at 30, 45, and 75% relative humidity
(RH) for 30 days. The samples were analyzed for the major degradation substances by
HPLC on day 0, 5, 10, and 30.
Analysis of Water Content
TGA and LOD were used to assess the water content of imipenem, cilastatin sodium,
and IMI/CIL. Karl Fischer titration was not suitable for the determination of the
water content of IMI/CIL because IMI/CIL contained sodium bicarbonate which interfered
with the measurement. These two methods were compared to identify and discuss the
most suitable method for providing valuable information.
TGA thermograms of imipenem and cilastatin sodium were recorded using the TGA Q500
V20.13 Build 39 thermoanalyzer apparatus (Lairui Instrument, Shanghai, China). The
weight loss of the samples was determined. The experiments were performed in alumina
crucibles and the samples were heated under a dynamic nitrogen atmosphere at a heating
rate of 10°C/min from 25 to 350°C with a sample mass of 5 to 10 mg.
For LOD assay, samples were dried in a vacuum oven (101–0ES, Keheng Co., Ltd., Shanghai,
China). The atmospheric pressure of the vacuum oven was set to below 2.67 kPa (20 mm
Hg). The temperature was set to 60°C and 100 g of samples were dried on a flat weighing
bottle for 3 hours until the weight of the samples stabilized (the difference between
the last two measurements was <0.02 g).
Drying Imipenem, Cilastatin Sodium, and IMI/CIL
The effect of decreasing the free water content in imipenem, cilastatin sodium, and
IMI/CIL on the stability of imipenem was evaluated. The free water in imipenem, cilastatin
sodium, and IMI/CIL was reduced by vacuum oven drying at 60°C. This experiment was
divided into the following three groups:
▪ Group 1: the dried and undried imipenem were filled into two vials, respectively,
and named L1 and L2.
▪ Group 2: the undried and dried cilastatin sodium were mixed with undried imipenem,
respectively, and named L3 and L4.
▪ Group 3: the dried and undried IMI/CIL were filled into two vials, respectively,
and named L5 and L6.
All the above vials filled with powder were purged with nitrogen to keep HO below
2%, and then sealed promptly. These samples were stored at 60°C and analyzed for the
major degradation substances by HPLC on day 0, day 5, day 10, and day 30.
Characterization of IMI/CIL from Different Batches
Imipenem, cilastatin sodium, and IMI/CIL from 2021–1, 2021–2, and 2021–3 were characterized
by X-ray powder diffraction (XRPD), polarized light microscopy (PLM), and laser diffraction
particle size distribution (PSD), and the relationship between physical properties
and stability of preparations were investigated.
XRPD Assay
The crystalline/amorphous nature of imipenem and cilastatin sodium in the solid state
(powder form) was estimated by an X-ray diffractometer (D8 Advance, Bruker AXS, Karlsruhe,
Germany). The patterns were collected in step scan mode at the rate of 0.02°/min.
Cu Kα radiation (1.5418 Å) was used at 40 kV and 40 mA and the 2-theta scan range
was set to 5°–60°. The diffraction data were analyzed with Origin 8.0 software (OriginLab,
Massachusetts, United States).
PLM Assay
The microscopic morphology of imipenem and cilastatin sodium was examined using a
XPN-990E polarized light microscope (Changfang Optical Instrument Co., LTD, Shanghai,
China). The imipenem and cilastatin sodium samples were sprinkled onto a glass slide
covered with a drop of dimethyl silicone oil to prevent exposure of the sample entities
to the atmosphere, and then covered with another glass coverslip. Observed samples
were determined to be in the crystalline form if they displayed uniform extinction,
and amorphous materials if they did not display uniform extinction. Images of samples
were collected using 100 × magnification, and TCapture software (Pooher Optoelectronics
Technology Co., LTD. Shanghai, China) was used to obtain the measurements.
PSD Analysis
Imipenem, cilastatin sodium, and IMI/CIL were characterized by a TOPSIZER laser diffraction
particle size analyzer (OMEC Instruments Co., Ltd, Zhuhai, China). PSDs were calculated
on a volume basis following the Mie theory.[7 ] The particle refractive index and particle absorption index used were 1.52 and 0.1,
respectively. All the measured particle diameters represent the area-equivalent or
volume-equivalent spherical diameter based on the technique of size measurement.
Statistical Analysis
Data were presented as mean of at least three trials, and value was recorded as mean ± standard
error of the mean. To evaluate the statistical significance of the differences between
groups, Student's t -test or one-way analysis of variance (ANOVA) was performed by using Excel (Microsoft
Office 2016, United States). p < 0.05 level of significance was used for all analyses.
Results and Discussion
Degradation Products
Imipenem and cilastatin sodium could be degraded into many impurities in the stability
studies. Three major degradation products of cilastatin sodium were identified as
(Z )-7-[(RS )-[(2R )-2-amino-2-carboxyethyl]sulfinyl]-2-[[[(1S )-2,2-dimethylcyclopropyl]carbonyl]amino]hept-2-enoic acid ([Scheme 1 ], Cil-A1), epimer ([Scheme 1 ], Cil-A2) at S, and (E )-(2RS )-7-[[(2R )-2-amino-2-carboxyethyl]sulfanyl]-2-[[[(1S )-2,2-dimethylcyclopropyl]carbonyl]amino]hept-3-enoic acid ([Scheme 1 ], Cil-G). Cil-A1 and Cil-A2 are oxidative degradation products. The main impurities
degraded by imipenem were imipenemoic acid ([Scheme 2 ], Imi-B1) and epimer ([Scheme 2 ], Imi-B2), which are hydrolytic degradation products. The above five impurities are
the focus of this study.
Scheme 1 Proposed degradation mechanism of cilastatin sodium.
Scheme 2 Proposed degradation mechanism of imipenem.
HO Levels
Stability of IMI/CIL at Different HO Levels
The effect of oxygen on the stability of IMI/CIL is shown in [Fig. 1 ]. After storing at 60°C for 30 days, the total impurities of samples under 2, 5,
and 20% of HO were 0.93 ± 0.02%, 1.75 ± 0.26%, and 2.71 ± 0.19%, respectively. The
degradation was significantly inhibited when HO was reduced (p < 0.05). This result indicated that HO has a significant effect on the degradation
of IMI/CIL. Among the many degradation impurities of IMI/CIL, three impurities Cil-A1,
Cil-A2, and Cil-G were identified to vary significantly with HO levels (p < 0.05). Impurities Cil-A1 and Cil-A2 are a pair of diastereoisomers, which are oxidative
degradation products of cilastatin sodium. According to the molecular structures of
the three impurities, the oxygen-mediated degradation mechanism of cilastatin sodium
was speculated as shown in [Scheme 1 ]. Cilastatin sodium contains a sulfur atom, which makes it easy to oxidize. When
cilastatin sodium is in an aerobic environment, the lone pair of electrons on the
oxygen atom acts as a nucleophile to oxidize cilastatin sodium to produce Cil-A1 and
Cil-A2. Cil-G is derived from the double bound migration of cilastatin sodium. Those
results suggested that oxygen is involved in the degradation of cilastatin sodium.
The stability of IMI/CIL can be greatly improved by controlling HO with nitrogen purging.
Fig. 1 Effect of HO levels on the stability of IMI/CIL from 2021–1 induced by stress condition
at 60°C. (A) Increase in total impurity content (%). (B) Increase in Cil-A1 content (%). (C) Increase in Cil-A2 content (%). (D) Increase in Cil-G content (%). Cil-A1, cilastatin-impurity A1; Cil-A2, cilastatin-impurity
A2; Cil-G, cilastatin-impurity G.
Stability of IMI/CIL from Different Batches at the Same HO Level
As shown in [Fig. 2 ], the degradation rate of IMI/CIL from 2021–1 and 2021–2 was still higher than that
from 2021–3 (p < 0.05) when the HO level of IMI/CIL was controlled at around 2% and stored at 60°C
for 30 days. This result indicated that the stability of IMI/CIL is affected not only
by HO levels, but also by other factors. It was found that two major impurities Imi-B1
and Imi-B2 increased faster in IMI/CIL from 2021–1 and 2021–2 than those from 2021–3
(p < 0.05), as shown in [Fig. 3 ]. Imi-B1 and Imi-B2 are a pair of diastereoisomers and hydrolytic degradation products
of imipenem. Imipenem contains a β-lactam ring that is particularly sensitive to water.
Many studies have shown that the degradation of β-lactam ring is related to water.[8 ]
[9 ] The β-lactam ring could be affected by OH- and H+ when it was exposed to water, resulting in the rupture of the amide bond on the quaternary
ring.[10 ] The hydrolytic degradation mechanism of imipenem is related to the opening of the
quaternary ring as shown in [Scheme 2 ].
Fig. 2 Stability of IMI/CIL from different batches induced by stress condition at 60°C for
30 days. (A) Increase in total impurity content (%). (B) Increase in Imi-B1 content (%). (C) Increase in Imi-B2 content (%). Imi-B1, imipenem-impurity B1; Imi-B2, imipenem-impurity
B2.
Fig. 3 Effect of RH on the stability of imipenem. (A-1) Decrease in imipenem content. (A-2) Partial enlarged view of (A-1). (B) Increase in Imi-B1 content (%). (C) Increase in Imi-B2 content (%). RH, relative humidity.
Effect of Water Content on Stability
Stability of Imipenem and Cilastatin at Different Relative Humidity
The stability of imipenem, cilastatin sodium, and IMI/CIL in different RH environments
is shown in [Figs. 3 ]
[4 ] to [5 ], respectively. As shown in [Fig. 3 ], after 30 days, the content of imipenem under RH 30, 45, and 75% were 98.07 ± 0.07%,
96.90 ± 0.2%, and 96.05 ± 0.03%, respectively. The higher the air humidity, the more
obvious the decrease of imipenem content (p < 0.05). This result confirmed that the stability of imipenem was indeed related
to water. Imi-B1 and Imi-B2 were the most significant growth impurities.
Fig. 4 Effect of RH on the stability of imipenem in 2021–1 preparation. (A) Decrease in imipenem content. (B) Increase in Imi-B1 content (%). (C) Increase in Imi-B2 content (%). RH, relative humidity; Imi-B1, imipenem-impurity
B1; Imi-B2, imipenem-impurity B2.
Fig. 5 Effect of RH on the content of cilastatin sodium in cilastatin sodium and IMI/CIL
from 2021–1. (A-1) Decrease in cilastatin sodium content in cilastatin sodium. (A-2) Partial enlarged view of (A-1). (B) Decrease in cilastatin sodium content in IMI/CIL. RH, relative humidity.
[Fig. 4A ] shows the stability of imipenem in IMI/CIL in different humidity environments after
30 days. Our data showed that the degradation of imipenem in IMI/CIL is more severe
than that in imipenem alone (p < 0.05). After 30 days, the imipenem content in IMI/CIL was close to null at 45%RH
and 75%RH, while the imipenem content in imipenem was still above 95%. These results
indicated that cilastatin sodium had a significant effect on imipenem under high humidity
(p < 0.05). Imi-B1 and Imi-B2 in IMI/CIL grew rapidly in the first 10 days, but decreased
dramatically from day 10 to day 30 (p < 0.05), and were further degraded to other impurities (note: there was no such decrease
in imipenem API at this time). Cilastatin sodium is an amorphous ingredient with hygroscopicity.
The moisture absorbed by cilastatin may have allowed enough water to be in contact
with crystalline imipenem under controlled RH conditions and induced its hydrolytic
degradation.
Another phenomenon observed in this study, as shown in [Fig. 5 ], is that imipenem could also affect the stability of cilastatin sodium. The degradation
of cilastatin sodium in IMI/CIL was more severe than that of cilastatin sodium alone
under controlled RH conditions (p < 0.05), indicating that the presence of imipenem also accelerated the degradation
of cilastatin sodium. Given above, water does have a significant effect on the stability
of imipenem and cilastatin sodium. Imipenem and cilastatin sodium in IMI/CIL are more
sensitive to moisture than individual imipenem or cilastatin sodium. Synergistic moisture
sorption and degradation occurred under controlled RH conditions.
Water Content of IMI/CIL
The total water contents of IMI/CIL from 2021–1, 2021–2, and 2021–3 are shown in [Table 2 ] and [Fig. 6 ]. The total water contents of samples measured by TGA are as follow: 2021–2 > 2021–3 > 2021–1.
However, as shown in [Fig. 3 ], IMI/CIL from 2021–3 with the higher total water content had the best stability,
rather than IMI/CIL from 2021–1 with the lowest total water content. The degradation
rate of IMI/CIL is consistent with the free water content of IMI/CIL measured in a
vacuum oven drying at 60°C as shown in [Table 3 ]: 2021–2 > 2021–1 > 2021–3 (p < 0.05). Total water content is a measurement of the total amount of water in samples,
which includes free water, adsorbed water, and bound water.[11 ] The absorbed and bound water has reduced energy and different properties than pure
water because it adsorbed on the drug surface or directly binds to the matrix through
hydrogen or ionic binding.[11 ] Adsorbed water and bound water are not detrimental to the stability of the drug.[12 ]
[13 ] Simply knowing the total water content may not be the most reliable method for understanding
the effects of water on drug stability. Unlike adsorbed and bound water, free water
has the same energy and properties as pure water, which is available for chemical
reactions. The rate of drug reactivity increases with increasing content of free water.
Consistent with this, this study further demonstrated that free water content is a
better predictor of the safety and stability of imipenem and cilastatin sodium than
total water content.
Table 2
TGA results
Sample
Batches
TGA weight loss (%)
IMI/CIL
2021–1
4.41
2021–2
4.84
2021–3
4.65
Imipenem
2021–1
5.39
Cilastatin sodium
2021–1
1.25
Abbreviation: TGA, thermogravimetric analysis.
Table 3
Results for vacuum drying at 60°C
Sample
Batches
Loss on drying (%)
IMI/CIL
2021–1
0.53 ± 0.05
2021–2
0.77 ± 0.04
2021–3
0.45 ± 0.05
Imipenem
2021–1
0.27 ± 0.03
Cilastatin sodium
2021–1
0.83 ± 0.07
Note: Each experiment was repeated three times.
Fig. 6 TGA results of IMI/CIL imipenem and cilastatin sodium. (A) IMI/CIL from 2021–1. (B) IMI/CIL from 2021–2. (C) IMI/CIL from 2021–3. (D) Imipenem of from 2021–1. (E) Cilastatin sodium from 2021–1. TGA, thermogravimetric analysis.
Drying Imipenem, Cilastatin Sodium, and IMI/CIL
The effects of reducing the free water content of imipenem, cilastatin sodium, and
IMI/CIL on the stability of imipenem were investigated. As shown in [Fig. 7 ], after storing at 60°C for 30 days, the total impurities in L1 and L2 were 3.09 ± 0.2%
and 2.77 ± 0.05%, respectively. The stability of L2 (dried) is better than that of
L1 (undried) (p < 0.05). Imi-B1 and Imi-B2 also decreased with the decrease of free water. This implies
that the degradation of imipenem can be effectively controlled by reducing the free
water content in imipenem. Although the free water content of undried imipenem was
only 0.27 ± 0.03%, the presence of trace free water can still affect the growth of
Imi-B1, Imi-B2, and total impurities (p < 0.05).
Fig. 7 Effect of reducing the free water content of imipenem on the stability of imipenem.
(A) Increase in total impurity content (%); (B) Increase in Imi-B1 content (%); (C) Increase in Imi-B2 content (%). Imi-B1, imipenem-impurity B1; Imi-B2, imipenem-impurity
B2.
As shown in [Fig. 8 ], the growth of Imi-B1 and Imi-B2 was significantly inhibited by drying cilastatin
sodium (p < 0.05). This result confirmed that free water in cilastatin sodium could also affect
the stability of imipenem, and the stability of imipenem could be improved by controlling
the free water content of cilastatin sodium. As previously discovered by Yoshioka
and Aso,[14 ] there is a phenomenon of water migration between different drug molecules. Cilastatin
sodium exists in an amorphous state in the preparation, and it is easy to absorb moisture.
Free water content of cilastatin sodium in IMI/CIL of 2021–1 was 0.83%, which was
the main source of free water in IMI/CIL. Our data suggested that the stability of
imipenem was affected by the intermolecular migration of water when imipenem was in
contact with cilastatin sodium.
Fig. 8 Effect of reducing the free water content of cilastatin sodium on the stability of
imipenem. (A) Increase in Imi-B1 content (%). (B) Increase in Imi-B2 content (%).
The effect of reducing the free water content of IMI/CIL on the stability of imipenem
was investigated. As shown in [Fig. 9 ], the stability of L6 was better than that of L5. The growth of total impurities,
Imi-B1, and Imi-B2 was inhibited after drying IMI/CIL (p < 0.05). This result showed that the degradation of imipenem in IMI/CIL could be
effectively prevented by reducing the free water content of IMI/CIL.
Fig. 9 Effect of reducing the free water content of preparation on the stability of imipenem.
(A) Increase in total impurity content (%). (B) Increase in imi-B1 content (%). (C) Increase in imi-B2 content (%).
Characterization of IMI/CIL from Different Batches
Result from XRPD Analysis
The XRPD patterns of imipenem and cilastatin sodium are shown in [Fig. 10A ] and [B ]. XRPD analysis of imipenem revealed the presence of the sharp diffraction peak signals
characteristic for the crystalline form, and the diffractogram of cilastatin sodium
displayed a broad amorphous halo with the absence of well-defined and intense peaks,
indicating that imipenem was crystalline in nature and cilastatin sodium was an amorphous
substance. As shown in [Fig. 10C ], it is clear that the XRPD pattern of IMI/CIL is almost a superposition of the patterns
contributed by imipenem and cilastatin sodium. The XRPD pattern of IMI/CIL from 2021–3
was consistent with that of 2021–1, indicating that the crystalline forms of the two
IMI/CIL were consistent.
Fig. 10 XRPD diffractograms of cilastatin sodium, imipenem, and IMI/CIL. (A) Cilastatin sodium. (B) Imipenem. (C) IMI/CIL from 2021–1. ( D) IMI/CIL from 2021–3. XRPD, X-ray powder diffraction.
Result from PLM Analysis
As drugs' properties strongly depend on the chemical and physical constitution, the
morphology of imipenem, cilastatin sodium, and IMI/CIL was explored with PLM. As shown
in [Fig. 11 ], a birefringence phenomenon was obviously observed in crystalline imipenem, whereas
no visible birefringence phenomenon was observed in amorphous cilastatin sodium. The
result was in a good agreement with the results obtained by XRPD. [Fig. 11E ] revealed that imipenem produced by 2021–3 is granular in shape and is relatively
uniform in size, neither elongated nor flat, and cilastatin sodium of 2021–3 is plate-shaped
and relatively thick, not elongated. As shown in [Fig. 11(A–C) ], imipenem from 2021–1 formed in an elongated, fiber-like and needle-like shape,
and cilastatin sodium was scale-like and angular and relatively thin. The form of
imipenem and cilastatin sodium from 2021–2 is similar to that of 2021–1, but the particle
size is much smaller. Hence, imipenem and cilastatin sodium from 2021–1 and B have
larger specific surface areas.
Fig. 11 Polarized microscopy images of imipenem, cilastatin sodium, and IMI/CIL. ( A) Imipenem of from 2021–1. ( B) Cilastatin sodium from 2021–1. ( C) IMI/CIL from 2021–1. ( D) IMI/CIL from 2021–2. ( E) IMI/CIL from 2021–3.
For drugs with different specific surface areas, the free water content and the number
of reactive groups exposed on the surface of drug particles are different. The larger
the specific surface area is, the higher free water content could be taken up and
the more reactive groups are exposed on the surface. In addition, another phenomenon
observed in this study is that large amounts of fine imipenem particles were adsorbed
on the surface of cilastatin sodium from 2021–1 and 2021–2, which was probably caused
by the electrostatic interaction. Consequently, the contact area between cilastatin
sodium and imipenem from 2021–1 and 2021–2 was greatly increased, which could result
in enhanced interaction between imipenem and cilastatin sodium. The adsorption kinetics
is related by energy barriers associated with electrostatic interactions. The larger
the specific surface area, the higher the free surface energy of cilastatin sodium;
the smaller the imipenem particles, the more imipenem particles adsorbed on cilastatin
sodium, thus, the greater the contact area between CIL and IMI. The water molecule
mobility often plays an important role in the degradation rate.[15 ] Materials that contain more moisture were capable of transferring it to other ingredients.[16 ] In addition, in water adsorption, the water taken up is dependent on the available
surface area.[16 ] The exposed surface of API particles is determined by the form of the drug.[17 ] Therefore, the larger the contact area between CIL and IMI, the more easily IMI
absorbs free water migrating from CIL, and then CIL has a greater influence on IMI.
This study confirmed that the micromeritic properties of drug particles, such as shape
and size, are of essential importance for the stability of drugs.
Result from Laser Diffraction Particle Size Analysis
[Fig. 12 ] illustrates the PSD curves of IMI/CIL from 2021–1, 2021–2, and 2021–3. It can be
seen that the PSD of IMI/CIL from 2021–1 was relatively wide, and the PSD of IMI/CIL
from 2021–3 was relatively narrow. Their largest volume fractions were both around
10 to 100 μm. However, due to the variability of particle shape, specific surface
area may be different. The IMI/CIL of 2021–1 might have a larger specific surface
area because imipenem of 2021–1 is elongated, fiber-like, and needle-like shape, and
cilastatin sodium was scale-like, angular, and relatively thin. The particle size
of IMI/CIL from 2021–2 was the smallest, mainly below 10 μm, and the specific surface
area was the largest. Therefore, the specific surface areas of preparations from 2021–1,
2021–2, and 2021–3 were in the order of 2021–2 > 2021–1 > 2021–3, which was correlated
well with the growth of Imi-B1 and Imi-B2 as shown in [Fig. 3 ].
Fig. 12 Particle size distribution of IMI/CIL from different batches. ( A) IMI/CIL of 2021–1. ( B) IMI/CIL of 2021–2. (C) IMI/CIL of 2021–3.
Conclusion
In this study, the effects of HO levels, water content, particle shape, and particle
size on the stability of IMI/CIL were investigated. Student's t -test and ANOVA were used to analyze the statistical significance of the differences
between groups. It was shown that reducing HO level by nitrogen purging could significantly
inhibit the degradation of cilastatin sodium. Oxygen caused some chemical reactions
of cilastatin sodium, such as oxidation reaction and double bond migration. It was
confirmed that moisture was detrimental to the stability of the imipenem. The higher
the RH, the more serious the degradation of imipenem. Compared with 2021–3 with a
higher total water content, the degradation of IMI/CIL was higher in 2021–1 with the
lowest total water content. Moreover, free water content was responsible for the degradation
of imipenem instead of the total water content. Free water in cilastatin sodium can
also affect the stability of imipenem through water molecule migration. Controlling
the free water content in imipenem or cilastatin sodium by vacuum oven drying can
effectively improve the stability of imipenem. The shape and size of imipenem and
cilastatin sodium particles also had a significant effect on the stability of IMI/CIL.
The greater the surface area of imipenem and cilastatin sodium exposed in the air,
the greater the effect of sodium cilastatin on imipenem. Based on the results obtained
above, limiting HO level, controlling the content of free water, as well as the shape
and size of imipenem and cilastatin sodium particles are highly recommended for the
production of IMI/CIL in the future.
