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Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgments
  7. REFERENCES

RATIONALE

Recent publications have reported that imatinib forms cyanide and methoxylamine adducts in vitro but without detail structural identification. The current work reports the identification of seven cyanide adducts that elucidate the bioactivation pathways and may provide hints for observed clinical adverse effects of the drug.

METHODS

Imatinib was incubated with human liver microsomal proteins in the presence of a NADPH-regeneration system and the trapping agents reduced GSH, potassium cyanide and methoxylamine. Samples were analyzed by high-performance liquid chromatography (HPLC) coupled with a LTQ-Orbitrap data collection system. Chemical structures were determined and/or postulated based on data-dependent high-resolution tandem mass spectrometric (MSn) exact mass measurements in both positive and negative scan modes, as well as in combination with hydrogen-deuterium exchange (HDX).

RESULTS

GSH and methoxylamine conjugates were either not detected or were in insufficient quantities for characterization. However, seven cyanide conjugates were identified, indicating that the piperazine and p-toluidine partial structures in imatinib can become bioactivated and subsequently trapped by the nucleophile cyanide ion. The reactive intermediates were postulated as imine and imine-carbonyl conjugate (α,β-unsaturated) structures on the piperazine ring, and imine-methide on the p-toluidine partial structure.

CONCLUSIONS

Chemical structures of seven cyanide adducts of imatinib have been identified or proposed based on high-resolution MS/MS data. Mechanisms for the formation of the conjugates were also proposed. The findings may help to understand the mechanism of hepatotoxicity of imatinib in humans. Copyright © 2013 John Wiley & Sons, Ltd.

Imatinib (Scheme 1) mesylate is a potent and relatively selective tyrosine kinase inhibitor that has been approved for treatment of Philadelphia-positive chronic myeloid leukemia (CML), acute lymphoblastic leukemia and advanced gastrointestinal stromal tumors.[1-3] Hepatotoxicity of imatinib has been reported, including transaminase and bilirubin elevation in patients[4-6] up to 5.1% and 3.1% in clinical trials, respectively. Several deaths were also reported from hepatic failure.[7, 8] However, the mechanism of the hepatotoxic response remains unelucidated. On the other hand, the formation of one or more reactive metabolites of imatinib is suggested by reports of time-dependent inactivation of CYP3A enzymes and by reports of formation of adducts of the drug with potassium cyanide and methoxylamine after incubations with human liver microsomes in vitro.[9-11] However, the latter report focused on the context of inactivation to cytochrome P450 enzymes and did not identify the chemical structures of the detected cyanide or methoxylamine conjugates. Hence, the bioactivation mechanisms of imatinib remain unexplained, and therefore which structural moieties become chemically reactive remains unknown. Since bioactivation is often speculated as responsible for clinical idiosyncratic toxicities,[12-14] including hepatotoxicity, we performed similar in vitro experiments but focused on the structural identification of seven cyanide conjugates that reveal the bioactivation pathways and may further help to elucidate the mechanism of hepatoxicity of imatinib in humans.

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Scheme 1. Chemical structure of imatinib.

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EXPERIMENTAL

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgments
  7. REFERENCES

Chemicals and reagents

Imatinib methanesulfonate salt was purchased from LC Laboratories (Woburn, MA, USA). Reduced GSH, potassium cyanide and methoxylamine hydrochloride were from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Ammonium acetate and HPLC-grade acetonitrile (ACN) and formic acid were all from Sigma-Aldrich or VWR International (West Chester, PA, USA). Purified water was obtained from an in-house Milli-Q ultrapurification system (Millipore, Billerica, CT, USA). Dideuterium monoxide (D2O) was purchased from Cambridge Isotope Laboratories, Inc. (Cambridge, MA, USA). Pooled male human liver microsomes (HLM) were purchased from Human Biologics International (Phoenix, AZ, USA).

Incubation and trapping of chemically reactive metabolites in vitro

Imatinib was incubated at 20 μM with 1.0 mg/mL pooled HLM protein, a NADPH-regenerating system, trapping agents (GSH and KCN at 1.0 mM and methoxylamine at 0.5 mM) and 50 mM Na/K phosphate buffer (pH 7.4) containing 3.3 mM MgCl2 and 1 mM EDTA. The NADPH-regenerating system consisted of separate solutions, as follows: Solution A – 25 mM NADP+, 66 mM glucose-6-phosphate and 66 mM MgCl2 in water, and Solution B – 40 U/mL of glucose-6-phosphate dehydrogenase in aqueous 5 mM sodium citrate added at 50 μL A to 10 μL B for a 1-mL reaction volume. The mixtures were incubated at 37°C in a shaking water bath for 60 or 120 min before the reactions were stopped by adding 3 volumes of ice-cold ACN followed by centrifugation (14000 g, 10 min and 4°C). The supernatants were removed, evaporated and reconstituted in ACN/water/formic acid (5:95:0.1, v/v/v; i.e., the starting HPLC mobile phase) for LC/MS analysis.

LC/MS/MS methods

The LC/MS/MS system consisted of a Shimadzu Promenence HPLC system (Kyoto, Japan) and an LTQ-Orbitrap mass spectrometer (Thermo Scientific, Somerset, NJ, USA) with an electrospray ionization (ESI) interface. The chromatography was performed on a Varian Polaris 3 C-18A column (150 × 2.0 mm, 3 µm particle size, Agilent, Palo Alto, CA, USA) coupled with a guard cartridge (4 × 3 mm; Phenomenex, Palo Alto, CA, USA). Samples were analyzed in both positive and negative modes using the same column, but the chromatography was conducted with slightly different mobile phases. For positive mode analysis, the analytes were eluted from the column with 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B). The stepwise linear gradient was 5% B (0–4 min), 30% B (4–33 min), 90% B (33–35 min), 90% B isocratic (35–39 min) and 5% B (39–40 min). For negative mode analysis, the HPLC mobile phases were 5 mM ammonium acetate in water (A) and ACN (B). The stepwise linear gradient was 10% B (0–4 min), 45% B (4–33 min), 90% B (33–35 min), 90% B isocratic (35–39 min) and 10% B (39–40 min). In both analysis modes, the column was re-equilibrated for 5 min prior to the next injection; hence, the total run was 45 min. The total mobile phase flow rate was set at 0.3 mL/min, and the column temperature was controlled at 35°C. For hydrogen-deuterium exchange (HDX) runs, D2O was used to replace H2O in the preparation of mobile phase A.

The mass spectrometer was tuned to optimal conditions for imatinib and was operated in a data-dependent acquisition mode, which consisted of a survey full scan (250–1000 m/z) at a resolution of 30 K and up to four dependent product ion scans at a resolution of 7.5 K. The product ions were generated in both collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) modes and were detected with the Orbitrap. HCD mode fragmentation was employed to detect low-mass ions, overcoming the low mass cutoff disadvantage of the CID mode.

Samples analyzed

One adduct of methoxylamine (M551) was detected in preliminary analysis in full scan mode but at such a low level that it was not suitable for MS/MS scans for structural elucidation. No GSH adduct was detected. Multiple cyanide adducts were detected, and these form the major focus of the current study.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgments
  7. REFERENCES

Metabolic profiling of imatinib in vitro and in vivo has largely focused on the products formed via N-dealkylation, N-oxidation, hydroxylation, and conjugation, which represent the bulk of the imatinib-derived materials.[15, 16] In contrast, the seven detected cyanide adducts obtained in the current study are at low levels, based on the full scan mass ion intensities (Fig. 1). In addition, Peak-1 and Peak-2 disappeared after several hours of storage on the HPLC sample tray (8°C).

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Figure 1. Extracted ion chromatograms of cyanide conjugates in comparison with mono-oxygenated and demethylated metabolites of imatinib.

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Identification of cyanide adduct M519B of imatinib

M519B was detected at both m/z 519.2607 (mono-protonated) and m/z 260.1342 (di-protonated) in full scan mode. The exact mass measurements indicated the conjugate was the net product of substitution of a hydrogen atom in imatinib by cyanide, without other structural modifications. Upon fragmentation of m/z 519 (CID, Fig. 2(A)), the mono-protonated molecule underwent an immediate elimination of a molecule of hydrogen cyanide and formed product ion m/z 492, along with other product ions. Compared with the fragment ions of imatinib (not shown) that were well characterized previously,[15, 16] the product ion m/z 394 suggested that the partial structures of the A, B, C and D ring systems were not modified in the adduct. This led to the assignment of the addition of the cyano group to the piperazine ring, which was consistent with the other two product ions at m/z 98 and 215. However, the exact position of the modification in the adduct still remained ambiguous. On fragmentation of the di-protonated molecule of the conjugate at m/z 260 (HCD, Fig. 2(B)), the two product ions m/z 423 and 70 demonstrated the complementary structure of the adduct after the loss of hydrogen cyanide. The two product ions unambiguously showed the reverse Diels-Alder fragmentation that occurred on the piperazine ring (Scheme 2). Hence, the cyano group of the conjugate must be located on C-3 or the equivalent C-5 position, indicating that an iminium cation was formed during the initial fragmentation. This proposal was confirmed by a follow-up HDX experiment in which the ion m/z 423 increased 3 m/z units to m/z 426, representing two exchangeable hydrogen atoms and one proton charge, as proposed in Scheme 2, while the ion m/z 70 remained the same, since it did not have exchangeable hydrogen atoms. In negative mode, the de-protonated molecule of the conjugate (m/z 517.2458) fragmented and lost a molecule of hydrogen cyanide, and so formed product ion m/z 490 (Fig. 3). The latter ion further underwent the reverse Diels-Alder process and formed m/z 462 by loosing a unit of ethane (Scheme 3). This is because in negative mode, the double-bond formation due to the elimination of hydrogen cyanide had to be between the two carbon atoms. Therefore, the information observed in negative mode was also consistent with the structural assignment derived from positive ion mode fragmentation.

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Figure 2. Collision-induced dissociation (A) and higher energy collision-induced dissociation (B) of M519B in positive mode.

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Scheme 2. Proposed collision-induced dissociation of M519B in positive mode.

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Figure 3. Collision-induced dissociation of M519B in negative mode.

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Scheme 3. Proposed collision-induced dissociation of M519B in negative mode.

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Peaks-1 and -2 showed nearly identical product ion spectra to M519B, but were detected only in freshly generated samples. Both disappeared after several hours (overnight) in the HPLC autosampler at 8°C, as well as in storage at –20°C. They appear to be different conformations of M519B. A molecular model simulation showed that the π-electrons of the cyano group in M519B could have interactions with the phenyl π-electrons (Ring D) and even the carbonyl π-electrons, given the rotation of the C8–C7 and N1–C7 bonds as well as possible chair and boat variable conformations of the piperazine ring. As the temperature is reduced, all M519B conformations converge to the one of lowest energy state, and, hence, behave as one structure, which elutes at the retention time of 21.94 min.

Identification of cyanide adduct M505 of N-demethylated imatinib

The adduct detected at m/z 505.2452 (mono-protonated) and 253.1265 (di-protonated) in positive full scan mode was proposed to be a cyanide conjugate of N-demethylated imatinib. Upon fragmentation of m/z 505 (Fig. 4(A)), the mono-protonated molecule proved the desmethyl assignment by the presence of product ions m/z 201 and 84, compared with the methyl-containing fragments (m/z 215 and 98, respectively) in M519B. Similarly, the product ion m/z 394 also indicated that the partial structures of the A, B, C and D ring systems were not modified in the conjugate. Therefore, the structural modification of this adduct must have also occurred on the piperazine ring. Upon fragmentation of the di-protonated molecule of the metabolite m/z 253, the presence of the product ion (Fig. 4(B)) m/z 423 indicated that M505 also underwent the reverse Diels-Alder fragmentation although the presumed complimentary product ion m/z 56 was not observed, and the cyano group was also added to C-3 (or equivalent C-5) position. Negative mode fragmentation of the deprotonated molecule m/z 503.2301 (Fig. 5) further supported this assignment. The observed product ion m/z 421 clearly showed the loss of a molecule of N-methyleneethenamine from ion m/z 476 in the same reverse Diels-Alder dissociation mechanism. Hence, the double bond in m/z 476 due to the loss of hydrogen cyanide must be between N-4 and C-3 (C-5) (Scheme 4). Negative mode CID using HDX of the deuterated molecule m/z 505.2429 revealed the fragmentation pathway of m/z 505.2429 [RIGHTWARDS ARROW]m/z 477.2249 [RIGHTWARDS ARROW]m/z 422.1837, which further confirmed the structural assignment.

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Figure 4. Collision-induced dissociation (A) and higher energy collision-induced dissociation (B) of M505 in positive mode.

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Figure 5. Collision-induced dissociation of M505 in negative mode.

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Scheme 4. Proposed collision-induced dissociation of M505 in negative mode.

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Identification of cyanide adduct M519A of imatinib

A second direct adduct between imatinib and the cyanide ion, namely M519A, was also detected at both m/z 519.2609 (mono-protonated) and 260.1343 (di-protonated) in full scan mode. The mass measurements of the protonated molecule indicated that there was no other structural modification other than the cyanide addition. Upon fragmentation of m/z 519, the product ions m/z 99 and 217 (Fig. 6) clearly indicated that rings D and E remained unmodified. Therefore, the conjugation had to occur on partial structure of ring A, B or C. This was in accordance with the presence of product ions m/z 419 and 301. Further CID of the doubly charged molecule m/z 260 showed two fragmentation pathways of m/z 260 [RIGHTWARDS ARROW]m/z 420 [RIGHTWARDS ARROW]m/z 301 [RIGHTWARDS ARROW]m/z 274 and m/z 260 [RIGHTWARDS ARROW]m/z 380 [RIGHTWARDS ARROW]m/z 263 (Fig. 7). Both product ions m/z 274 and 263 seemed to indicate that the modification occurred on the C ring, but there were no solid data to rule out the possibility of cyanide addition to rings A or B. Without other modifications, such as hydroxylation, that introduces electron-withdrawing potential, it is hard to rationalize electrophilic addition to ring A (pyridine) or B (pyrimidine), for which no publications reporting bioactivation were identified. Therefore, the addition of the cyano group was proposed to be on the p-toluidine (ring C) partial structure. This partial structure has been reported to become bioactivated via an imine-methide intermediate and proven to be electrophilic.[17, 18] Hence, M519A was proposed as the result of Michael addition of the cyanide ion to the methyl carbon (C-31) through a hypothesized imine-methide intermediate. The latter could be formed from dehydration of a hydroxylated metabolite with the hydroxylation on the methyl carbon atom (C-31). This hydroxylated metabolite was reported in previous work and was also detected and identified in the current experiments.[12, 13] The mass fragmentation pathway of M519A is proposed in Scheme 5.

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Figure 6. Collision-induced dissociation of M519A in positive mode.

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Figure 7. Further collision-induced dissociation of M519A in positive mode.

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Scheme 5. Proposed collision-induced dissociation of M519A in positive mode.

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Identification of cyanide adducts M519C to M519F of imatinib

As shown in Fig. 1, M519C–M519F were also detected in full scan MS. Their protonated molecules (m/z 519.2244, 519.2245, 519.2243 and 519.2246, respectively) led to the conclusion that they were cyanide conjugates of demethylated imatinib with an additional carbonyl functionality, based on their smaller mass defects compared with the corresponding molecules of M519A and M519B. This conclusion is consistent with the HDX result, which showed that all four conjugates increased by 4 m/z units, proving that they all had three exchangeable hydrogen atoms in addition to the charge. Upon HCD (Fig. 8), all four conjugates generated the common product ion m/z 394, indicating that the partial structures of the A, B, C and D ring systems were not altered (Scheme 6). Therefore, the structural modifications must all be on the piperazine ring. This conclusion is confirmed by the product ions of m/z 215 (M519C, M519D and M519F) and 119 (M519D, M519E and M519F). For all four positional isomers, determination of the exact structure for each of the chromatographic peaks would require chemical synthesis, but should not be necessary for the conclusion that a bioactivation reaction has occurred. The addition of the cyano group on the piperazine ring introduced a chiral center; therefore, each of the conjugates may be a mixture of two enantiomers, not separable on non-chiral HPLC columns.

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Figure 8. Higher energy collision induced dissociation of M519C–M519F in positive mode.

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Scheme 6. Proposed fragmentation of M519C–M519F.

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Proposal of the methoxylamine adduct M551 of imatinib

As mentioned before, the methoxylamine adduct was detected at both m/z 551.2517 (mono-protonated) and 276.1295 (di-protonated) in full scan mode at such a low level that a quality product ion spectrum could not be collected. These data correspond to elemental compositions of C30H31N8O3+ (m/z 551.2514) and C30H32N8O32+ (m/z 276.1293) with merely 0.5 and 0.7 ppm measurement biases, respectively. Hence, the adduct is proposed as imatinib incorporated with methoxylamine in addition to one double bond and one carbonyl functionality. While the latter two functionalities could be located on the piperazine moiety, including C-7, the exact position of the methoxylamine remains unknown. Yet, given the identification of M505 and M519A to M519F, the incorporation of methoxylamine could be speculated to be on the piperazine or p-toluidine moiety as well.

Bioactivation mechanism

With the structural identification of all seven cyanide adducts, the bioactivation pathways of imatinib can be proposed as shown in Scheme 7. Both the piperazine and the p-toluidine moieties undergo P450-catalyzed oxidation and subsequent desaturation (dehydration or dehydrogenation), forming imine and imine-methide intermediate structures, respectively. Hydroxylation on C-31 of the p-toluidine was previously reported. Although hydroxylation on the piperazine ring has not been previously observed, the reported lactam formation must be a subsequent dehydrogenation result of an initial hydroxylation on the carbon atoms.[15, 16]

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Scheme 7. Proposed bioactivation mechanisms of imatinib.

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The piperazine ring moiety is a common substructure for pharmaceuticals as it is present in more than 200 marketed drugs.[19] Upon metabolism, it typically undergoes several well-known biotransformations, such as amide formation (via initial hydroxylation on an adjacent carbon atom), N-dealkylation (leading to ring opening) and N-oxidation. Additional less frequent metabolic pathways have also been reported, including N-hydroxylation, N-glucuronidation, N-sulfonation and formation of a carbamoyl glucuronide. Although these biotransformation reactions are not typically associated with reactive intermediates, there are reports describing the identification of metabolites formed due to electrophilic addition with nucleophilic 'trapping' agents (GSH, KCN, methoxylamine, etc). Doss et al.[20] and Gu et al.[21] reported that the piperazine substructure in their drug candidates formed conjugated imine (α,β-unsaturated) substructures that were trapped with GSH and subsequently underwent a ring-contraction rearrangement. An earlier report by Kalgutkar et al.[22] described metabolic activation of a drug candidate due to the formation of imine or nitrone intermediates from the piperazine ring substructure. All previous and current findings with regard to bioactivation of a piperazine ring have been proposed to be the result of oxidative desaturation, probably through direct two-electron oxidation and hydroxylation/dehydration processes, on the piperazine moiety, to form imine or conjugated imine intermediates.

The proposed imine-methide intermediate formed on the p-toluidine moiety should be a typical soft electrophile, given that the valence electrons can be easily delocalized and conjugated with an electron-withdrawing carbonyl (C-15) group. In theory, it should easily react with a soft nucleophile such as GSH, as shown in previous reports.[17, 18, 23] However, in the experiments reported herein, it only reacted with cyanide ion, which is a hard nucleophile, instead of GSH. This is postulated to have been due to the steric effect caused by the adjacent large pyridine-pyrimidine partial structure. The smaller cyanide ion was thus able to attack the electrophile, although the formation of this derivative was at a low level nevertheless (Fig. 1) judging by MS intensities. Also noteworthy is the formation of M519C–M519F. With a carbonyl group on the ring, it would be highly likely to form conjugated imine (α,β-unsaturated) intermediates, such as imine amides[24] or similar structures to the reported reactive species,[20, 21] which are also soft electrophiles. However, conjugation with the soft nucleophile GSH was not detected in the current work. This finding seems to imply that the cyanide adducts (M519C–M519F) were not formed via a conjugated imine intermediate. Yet, given the numbers (4) of positional isomers formed on a six-membered ring, the possibility of a conjugated imine intermediate cannot be ruled out.

Bioactivation in relation to observed clinical adverse effects

Although acute liver failure has been reported as 'rare' in clinical use of imatinib, the drug caused transaminase elevation in up to 5.1% and bilirubin elevation in up to 3.5% of patients in clinical trials, both clinical events indicative of liver toxicity. To the authors' knowledge, there has been no previous investigation into the hepatotoxicity mechanism of imatinib reported thus far. The current findings of potentially reactive intermediates of the drug may shed light on the causes of these observed adverse drug effects. For further new drug discovery and optimization with regards to piperazine moiety, attempts of substitution with alkyl groups on the moiety have shown promise in retaining pharmacological potency while reducing irreversible covalent protein binding.[25] The alkyl substitution on the piperazine moiety likely blocked or interrupted enzymatic oxidation/hydroxylation on the carbon atoms, hence blocking or reducing possible formation of an imine intermediate.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgments
  7. REFERENCES

We have described the structural determination of cyanide adducts of the selective kinase inhibitor imatinib in which the piperazine ring and p-toluidine substructures underwent bioactivation. We also proposed the mechanisms for the formation of the detected conjugates. This investigation echoes the previous reports of potential bioactivation of these two substructures in other drug candidates or chemical entities. The findings may allow a deeper understanding of the metabolism and toxicology of imatinib in humans.

Acknowledgments

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgments
  7. REFERENCES

The author greatly appreciates insightful discussions with Drs. Anthony Drager and Jian Chen of Teva Branded Pharmaceutical Products R&D, Inc. This research work was funded by Teva Branded Pharmaceutical Products R&D, Inc.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgments
  7. REFERENCES
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