Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2020 Sep 11;5(37):23885–23891. doi: 10.1021/acsomega.0c03121

Chiral Chromatographic Isolation on Milligram Scale of the Human African Trypanosomiasis Treatment d- and l-Eflornithine

Mikael Boberg †, Anna C Jonson ‡, Hanna Leek ‡, Rasmus Jansson-Löfmark †,§, Michael Ashton †,*
PMCID: PMC7513348  PMID: 32984708

Abstract

graphic file with name ao0c03121_0007.jpg

Eflornithine is a recommended treatment against the otherwise fatal parasitic disease late stage human African trypanosomiasis (HAT), also known as Gambian sleeping sickness. It is administered repeatedly as a racemic mixture intravenously (IV) together with oral nifurtimox. Racemic eflornithine has been investigated in clinical trials for oral dosing. However, due to low systemic exposures at a maximum tolerated oral dose, the drug is continued to be administered IV. The eflornithine enantiomers, d- and l-eflornithine, have different affinities to the target enzyme ornithine decarboxylase, suggesting that the pharmacodynamics of the enantiomers may differ. The aim of this study was to develop a method for isolation of d- and l-eflornithine from a racemic mixture. Several chiral stationary phases (CSPs) were evaluated for enantioselectivity using supercritical fluid chromatography (SFC) or high-performance liquid chromatography (HPLC). None of the tested CSPs rendered separation of the enantiomers in SFC mode. Separation of the enantiomers with SFC on the CSP Chiralpak IG was only achieved on an analytical scale after derivatization with ortho-phthalaldehyde (OPA). This was the first reported enantioselective SFC method for an eflornithine derivate. However, due to poor stability, the eflornithine-OPA derivates degraded and no chemically pure enantiomers were obtained. The CSP that showed enantioselectivity in HPLC mode was Chirobiotic R, which resulted in a successful isolation on a semipreparative milligram scale. The isolated eflornithine enantiomers will be tested in nonclinical in vitro and in vivo studies to support and assess the feasibility of a future clinical program with an oral HAT treatment.

Introduction

The neglected tropical disease human African trypanosomiasis (HAT), also known as sleeping sickness, threatens millions of people in sub-Saharan Africa.1 HAT is a fatal disease unless treated, and it has been estimated that over 13 million people live in areas with moderate to very high risk of being infected by Trypanosoma brucei (T.b.) gambiense or rhodesiense parasites.2,3 HAT has been ranked as the third most important parasitic disease when assessed by the disability-adjusted life years (DALY) lost.4 The anticipation from key stake holders like the World Health Organization (WHO) is that the incidence of gambiense HAT will decrease as a public health problem and be fully eliminated by 2030.5,6 This ambitious target needs resources to be achieved and sustained.7,8

The active pharmaceutical ingredient eflornithine (Ornidyl), also known as dl-α-difluoromethylornithine, is a recommended treatment in combination with nifurtimox (Lampit) for patients infected with T.b. gambiense that have reached the late stage of the disease. Both drugs are included in the WHO model list of essential medicines. The nifurtimox–eflornithine combination therapy has been reported to be superior to other drugs against HAT as well as to eflornithine or nifurtimox monotherapy.9−11 Eflornithine inhibits the enzyme ornithine decarboxylase (ODC) that is important for polyamine biosynthesis.12,13 Eflornithine is currently dosed intravenously (IV) as a racemic mixture. Racemic eflornithine for oral dosing has been investigated in clinical trials.14−28 However, due to poor systemic exposure at a maximum tolerated oral dose, the drug is continued to be administered IV. It has been shown that the d- and l-eflornithine enantiomers have different affinities to ODC in a cell free in vitro assay.12 This suggests that the enantiomers could exhibit different pharmacodynamics (PD) since the target affinity of l-eflornithine was superior to d-eflornithine. With an oral drug formulation of the potentially more potent l-eflornithine, it is conceivable that a therapeutic systemic exposure could be obtained at doses below the maximum tolerated oral dose of racemic eflornithine.

Within early drug discovery, chiral chromatography is a common and time-efficient technique to isolate enantiomers in high purity. Historically, high-performance liquid chromatography (HPLC) was the dominating technique.29 More recently, supercritical fluid chromatography (SFC) has been developed and can today be considered a gold standard for preparative chromatography of enantiomers in early drug discovery.30,31 A wide variety of chiral stationary phases (CSPs) with unique chemical characteristics are commercially available. Other complementary techniques, such as asymmetric synthesis, are often more time-consuming and yield only one of the enantiomers and are, therefore, considered less efficient in early drug discovery.29,32

Eflornithine is a chiral small zwitterionic compound (MW = 182 g/mol) lacking chromophores. Such physicochemical properties are disadvantageous for chiral recognition since enantioselectivity is harder to achieve with a limited number of interaction points via, for instance, hydrogen bonds, van der Waals forces, π–π interactions, and/or steric hindrance. The eflornithine enantiomers have previously been separated and quantified in biological fluids on a Chirobiotic TAG column33 and on a bioanalytical scale using derivatization or with chiral eluant.34−37 In the present study, the Chirobiotic TAG method was used to identify the elution order of d- and l-enantiomers and also evaluate on a semipreparative milligram scale. Evaporative light scattering (ELS) or mass spectrometry (MS) detection have been used to circumvent the lack of chromophores. In the present study, eflornithine was derivatized with ortho-phthalaldehyde (OPA)34,38−41 to enhance the detectability and to allow increased chiral recognition by the CSP. Furthermore, we evaluated the opportunity to isolate an eflornithine-OPA derivate using chiral chromatography in SFC mode.

The aim of this study was to develop a method to isolate pure d- and l-eflornithine from a racemic mixture on a milligram scale using chiral chromatography. This work has high potential clinical importance since enantiopure eflornithine has never been dosed in a clinical setting. The more active l-eflornithine enantiomer could be used in key nonclinical in vitro and in vivo studies to support decision making whether a future clinical program with oral HAT treatment is feasible.

Results and Discussion

Chiral Chromatographic Isolation of d- and l-Eflornithine

Enantioselectivity for eflornithine (Figure 1a) has previously been reported using HPLC on an analytical scale with the Chirobiotic TAG column.33 This method was considered unsuitable for a semipreparative scale isolation due to the long retention time and high water content in the mobile phase. Today, more than 80% of the racemic compounds in early drug discovery at AstraZeneca R&D, Sweden are separated by SFC.32 Since this is often a fast and cost-efficient approach to obtain pure enantiomers, we aimed to identify a method using this technique. Although a wide variety of CSP and modifier combinations were evaluated in SFC mode, none of them showed enantioselectivity for d- and l-eflornithine (Supplementary Table S1). Therefore, HPLC mode was used for further studies. However, it turned out to be difficult to identify a suitable method for the isolation of d- and l-eflornithine also in this mode (Supplementary Table S2). The only CSP, apart from the previously identified Chirobiotic TAG method, that showed enantioselectivity was Chirobiotic R. Therefore, this CSP was selected for further method optimization. The selectivity and retention of the eflornithine enantiomers were evaluated by varying the acid–base ratio, total ionic strength, and polarity of the mobile phase. The best method obtained with a selectivity (α) of 1.2 was with methanol (MeOH):H2O 90:10 (v/v%) containing 175 mM acetic acid (AcOH) and 35 mM triethylamine p.a. (TEA) (Figure 1b).

Figure 1.

Figure 1

Open in a new tab

(a) Structure of the zwitterionic eflornithine molecule. The asterisk indicates the chiral center. (b) Chiral separation of d- and l-eflornithine with Chirobiotic R (4.6 × 250 mm, 5 μm) with a mobile phase of MeOH:H2O 90:10 (v/v%) with 175 mM AcOH and 35 mM TEA, 40 °C, with a flow rate of 1 mL/min, a sample concentration of 1 mg/mL in MeOH, and detection was made with MS.

Chiral Chromatographic Isolation of Eflornithine-OPA Derivate

Since no enantioselectivity was obtained for eflornithine in SFC mode, an OPA derivate of eflornithine was investigated as alternative means to achieve enantioselective separation. Different CSP and modifier combinations were evaluated (Supplementary Table S1). The best method was identified using Chiralpak IG with 35% ethanol (EtOH) containing 20 mM ammonia (NH3) as a modifier in CO2 (Figure 2a). The selectivity (α) for the final analytical SFC method was 1.3. This method was then applied on a semipreparative scale where the two enantiomer derivates were fractionally collected (Figure 2b). However, due to low stability, the separated OPA derivates both degraded during the chromatography and evaporation procedures and no chemically pure enantiomers were obtained. Moreover, stability issues with the eflornithine-OPA derivate have previously been reported.38,42,43 Still, the developed method is, to the best of our knowledge, the first reported enantioselective SFC method for an eflornithine derivate. This 5 min method was used throughout the study for quick initial determination of enantiomeric purity of isolated nonderivatized d- and l-eflornithine obtained in HPLC mode. The SFC method was compatible with different types of detectors such as MS, ELS, and ultraviolet–visible (UV) spectroscopy. Moreover, it could be used as a starting point for bioanalytical method development and subsequent validation.

Figure 2.

Figure 2

Open in a new tab

(a) Analytical method for d- and l-eflornithine derivatized with OPA separated in SFC mode using Chiralpak IG (4.6 × 150 mm, 3 μm) with a mobile phase of 35% EtOH containing 20 mM NH3 in CO2, at 120 bar, 40 °C, with a flow rate of 3.5 mL/min and detection at 240 nm. (b) Semipreparative method on Chiralpak IG (20 × 250 mm, 5 μm) with 35% EtOH containing 20 mM NH3 in CO2, 40 °C, with a flow rate of 70 mg/min and detected at 240 nm.

Milligram Scale d- and l-Eflornithine Isolation on Chirobiotic R

Since the approach with the eflornithine-OPA derivate was unsuccessful, the novel optimized method on Chirobiotic R in HPLC mode was used for semipreparative isolation (Figure 3). Each cycle, 10 mg of racemic eflornithine was injected. The first eluted peak was collected separately and subsequently identified as a solvent peak related to neither d- nor l-eflornithine.

Figure 3.

Figure 3

Open in a new tab

Milligram scale isolation of d- and l-eflornithine in HPLC mode using Chirobiotic R (20 × 250 mm, 5 μm) and MeOH:H2O 90:10 (v/v%) containing 175 mM AcOH and 35 mM TEA at ambient temperature with a flow rate of 18 mL/min. The racemic sample (10 mg; 30 mg/mL (aq)) was injected and detected with ELS. The peak in front of the following two enantiomer peaks was identified as a solvent peak.

The previously published analytical method on Chirobiotic TAG33 was used to identify the elution order of the isolated enantiomers from the semipreparative run. The preparative results showed the same elution order, i.e., with d-eflornithine as the first eluting enantiomer (Figure 4).

Figure 4.

Figure 4

Open in a new tab

Enantiomeric elution order was determined using the Chirobiotic TAG method published by Malm and Bergqvist33 (4.6 × 250 mm, 5 μm) with a mobile phase of buffer:EtOH 75:25 (v/v%) (buffer containing 17 mM AcOH and 2 mM TEA) at ambient temperature with a flow rate of 1 mL/min, and MS was used for detection.

To compare with the optimized method on Chirobiotic R, also, the throughput and loadability on the Chirobiotic TAG column were evaluated on a semipreparative scale (Supplementary Figure S1). Analytically, both methods showed similar selectivity (α = 1.2). However, on a semipreparative scale, a four-fold longer retention time and broader peaks were observed on the Chirobiotic TAG column. Moreover, the evaporation time of the collected fractions was also prolonged by a mobile phase containing over 7 times more water. Overall, despite similar amounts of racemic eflornithine injected, this method was inferior to isolation on Chirobiotic R. On this CSP, 160 mg of d-eflornithine and 157 mg of l-eflornithine were obtained after 65 injections in enantiomeric purities of 99.1 and 95.7%, respectively (Figure 5). Moreover, the enantiomeric purity results were confirmed by using a validated HPLC method38 and the acquired results were comparable. The recoveries for the enantiomers were 49% (160 mg of 325 mg) for d-eflornithine and 48% (157 mg of 325 mg) for l-eflornithine. The middle fraction could be collected and reinjected to improve the recovery, but this was not done in the present study since enough racemic material was available.

Figure 5.

Figure 5

Open in a new tab

Determination of enantiomeric purity in SFC mode using Chiralpak IG (4.6 × 150 mm, 3 μm) with a mobile phase of 35% EtOH containing 20 mM NH3 in CO2, at 120 bar, 40 °C, with a flow rate of 3.5 mL/min and detection at 240 nm. (a) d-eflornithine sample with an enantiomeric purity of 99.1% and (b) l-eflornithine sample with an enantiomeric purity of 95.7%.

Comparison of the Methods Developed in SFC and HPLC Mode

Chromatography for the developed SFC method for the eflornithine-OPA derivate in the present study was more efficient as compared to the HPLC method using Chirobiotic R. For example, the cycle time was shorter and less solvent was used. This would decrease the evaporation time needed for isolation. However, the poor stability of the d- and l-eflornithine-OPA derivates38,42,43 was a negative factor and resulted in no pure enantiomers. It was, as expected, difficult to find a CSP that would render chiral separation of d- and l-eflornithine in SFC or HPLC mode. However, it was possible to obtain enantioselectivity in HPLC mode as Chirobiotic R and TAG with the chiral antibiotic macromolecules were able to interact more with the eflornithine enantiomers when comparing to the other CSPs.

Study Limitations

The number of studied CSPs for racemic eflornithine was 14 in SFC and HPLC mode with 32 and 31 CSP and modifier combinations. Other derivatization agents than OPA could be investigated to explore the possibilities to acquire chiral isolation of derivatized eflornithine. On the other hand, the stability of derivatized enantiomers and, in addition, the removal step of the derivatization agent after the chiral separation may also be challenging. These limitations mentioned above provide opportunities for further extensive studies. Additionally, there are also recent publications on the direct resolution of amino acids using SFC and HPLC.44,45 These approaches may be useful for any future work. However, such studies are beyond the scope of the present study.

Importance of Enantiomer Specific Interpretation in Drug Development

In general, the knowledge around chirality and enantiomer pharmacokinetics (PK), PD, and toxicology profiles has expanded over the years where omeprazole and verapamil are two out of numerous examples with enantioselective PK or PD.46−50 Moreover, the imperative safety and toxicity aspects must be taken into account for different enantiomers where the thalidomide disaster is a historical landmark.51 Also, guidelines from regulatory agencies require PK and toxicology data for both the racemic mixture and enantiomers.52,53 For eflornithine, oral dosing of the racemic mixture has been studied both in nonclinical35,54−57 and clinical14−28,58 settings. More recently, the synthesis of N-acetylated amine or α-ethyl ester prodrugs59,60 represents an interesting approach for the development of an oral eflornithine treatment. However, the benefit of dosing the individual eflornithine enantiomers against gambiense HAT still remains to be investigated and is enabled by the identified HPLC method using Chirobiotic R.

Conclusions

This is the first milligram scale isolation of the d- and l-enantiomers of the zwitterionic compound eflornithine using chiral semipreparative HPLC. The d- and l-enantiomers of eflornithine were isolated using Chirobiotic R. A wide variety of chiral stationary phases and modifiers were investigated in SFC mode, and even though the chiral stationary phases have unique chemical features, none exhibited enantioselectivity for d- and l-eflornithine. In addition, the first enantioselective 5 min SFC method for the eflornithine-OPA derivate was developed on an analytical scale. The isolated eflornithine enantiomers will be tested in nonclinical in vitro and in vivo studies to support and assess the feasibility of a future clinical program with an oral HAT treatment.

Experimental Section

Chemicals

Eflornithine hydrochloride was kindly donated by the WHO/World Bank Tropical Disease Research (Geneva, Switzerland). OPA was purchased from Sigma-Aldrich (St. Louis, MO, USA). All HPLC grade solvents were obtained from Sigma-Aldrich (Seelze, Germany), except for EtOH (99.5%), which was obtained from UnivarSolutions (Haninge, Sweden). Liquid carbon dioxide was purchased from AGA Gas AB (Stenungsund, Sweden). AcOH was purchased from Riedel-de Haën (Seelze, Germany), diethylamine p.a. (DEA) and TEA were from Fluka (Buchs, Switzerland), and NH3 was from ThermoFisher (Kandel, Germany).

Columns and Modifiers

The CSPs used in SFC mode were Chiralpak (IA, IB, IB N-3, IC, ID, IE, IG, AD, and AS), Chiralcel OJ, and tert-butylcarbamoylquinine (tBuCQN) from Chiral Technologies (Illkirch, France); Lux Amylose 1 and Lux Cellulose 3 and 4 from Phenomenex (Torrance, CA, USA); (S,S)Whelk-O1 from Regis Technologies (Morton Grove, IL, USA); Kromasil CelluCoat from Eka Chemicals (Bohus, Sweden); Chiralart Cellulose SJ from YMC (Kyoto, Japan); and Chirobiotic (T and V2) from Astec (Whippany, NJ, USA). The dimensions and particle sizes for the columns were 150 mm × 4.6 mm I.D. and 3 μm in SFC mode and 250 mm × 4.6 mm I.D. and 5 μm in HPLC mode. The CSPs used for the HPLC evaluation were Reprosil Chiral-NR from Dr. Maisch (Ammerbuch, Germany), Chiralpak (IA, IB, IC, ID, and AD), tBuCQN, (S,S)Whelk-O1, and Chirobiotic (T, R, V, V2, T2, and TAG) from Astec (Whippany, NJ, USA). The organic modifiers MeOH, EtOH, and isopropyl alcohol (2-PrOH) were used in SFC mode with an initial gradient of a 5–40% modifier with and without additives. Mobile phases based on MS and ELS compatible solvents (MeOH, EtOH, 2-PrOH, and acetonitrile (ACN)) with additives were evaluated using isocratic elution in HPLC mode.

Chromatographic Systems

The analytical SFC runs were performed on an Acquity UPC2 instrument equipped with a photodiode array detector and a single quadrupole detector using the software Empower (version 3, Waters, Milford, MA, USA). The flow rate was 3.5 mL/min, the back pressure was set to 120 bar, and the column temperature was 40 °C. The preparative SFC system used was Supersep 150 equipped with a UV spectroscopy detector using the software Proficy HMI/SCADA iFIX (version 5.1, Novasep, Pompey, France).

The analytical HPLC runs were performed on Alliance 2695 equipped with a Waters Micromass ZQ detector (Waters, Milford, USA) using the software MassLynx (version 4.1). The flow rate was 1 mL/min, and the initial column temperature was 25 °C. The semipreparative HPLC system used was an Interchim puriFlash 4250 integrated with a puriFlash AS-I sampler, UV, ELS, and MS detectors using the software Interchim soft (V5.1c.09, Montluçon, Cedex, France). The collected d- and l-eflornithine fractions were evaporated using a Rotavapor R II rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) and a Biotage V-10 evaporator system (Biotage AB, Uppsala, Sweden). To identify the elution order of the enantiomers, Chirobiotic TAG (4.6 × 250 mm, 5 μm) with a mobile phase of buffer:EtOH 75:25 (v/v%) (buffer containing 17 mM AcOH and 2 mM TEA) was used.33

Acknowledgments

The authors wish to thank Marcus Malmgren for his input with regard to the chemistry of eflornithine. We also would like to thank Linda Thunberg, Anna-Carin Carlsson, and Kurt-Jürgen Hoffmann for their invaluable reviews of the manuscript. The Swedish Research Council funded the project (2016-05780). Although co-authors A.C.J., H.L., and R.J.-L. are employed by AstraZeneca R&D in Gothenburg, Sweden, AstraZeneca contributed in kind and had no influence over the study.

Glossary

Abbreviations

human African trypanosomiasis

(HAT)

supercritical fluid chromatography

(SFC)

high-performance liquid chromatography

(HPLC)

ultraviolet–visible spectroscopy

(UV)

mass spectrometry

(MS)

evaporative light scattering

(ELS)

chiral stationary phase

(CSP)

pharmacokinetics

(PK)

pharmacodynamics

(PD)

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03121.

  • Milligram scale HPLC isolation of d- and l-eflornithine using Chirobiotic TAG (20 × 250 mm, 5 μm) with buffer:EtOH 75:25 (v/v%) (buffer containing 17 mM AcOH and 2 mM TEA) at ambient temperature, 18 mL/min with 10 mg of the racemic sample (30 mg/mL (aq)) injected and detected with ELSD (Supplementary Figure S1); chiral stationary phase and modifier combinations evaluated for d- and l-eflornithine and the eflornithine-OPA derivate in SFC mode (Supplementary Table S1); and chiral stationary phase and modifier combinations evaluated for d- and l-eflornithine in HPLC mode (Supplementary Table S2) (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c03121_si_001.pdf (212.6KB, pdf)

References

  1. Aksoy S.; Buscher P.; Lehane M.; Solano P.; Van Den Abbeele J. Human African trypanosomiasis control: Achievements and challenges. PLoS Neglected Trop. Dis. 2017, 11, e0005454 10.1371/journal.pntd.0005454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Simarro P. P.; Cecchi G.; Franco J. R.; Paone M.; Diarra A.; Priotto G.; Mattioli R. C.; Jannin J. G. Monitoring the Progress towards the Elimination of Gambiense Human African Trypanosomiasis. PLoS Neglected Trop. Dis. 2015, 9, e0003785 10.1371/journal.pntd.0003785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Kennedy P. G. E. Human African trypanosomiasis: in and out of Africa. Neurology 2006, 66, 962–963. 10.1212/01.wnl.0000208221.55385.55. [DOI] [PubMed] [Google Scholar]
  4. Kennedy P. G. E. The continuing problem of human African trypanosomiasis (sleeping sickness). Ann. Neurol. 2008, 64, 116–126. 10.1002/ana.21429. [DOI] [PubMed] [Google Scholar]
  5. WHO WHO Executive Summary ;2012.
  6. WHO WHO interim guidelines for the treatment of gambiense human African trypanosomiasis. WHO interim guidelines for the treatment of gambiense human African trypanosomiasis; 2019. [PubMed]
  7. Akazue P. I.; Ebiloma G. U.; Ajibola O.; Isaac C.; Onyekwelu K.; Ezeh C. O.; Eze A. A. Sustainable Elimination (Zero Cases) of Sleeping Sickness: How Far Are We from Achieving This Goal?. Pathogens 2019, 8, 135. 10.3390/pathogens8030135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franco J. R.; Cecchi G.; Priotto G.; Paone M.; Diarra A.; Grout L.; Simarro P. P.; Zhao W.; Argaw D. Monitoring the elimination of human African trypanosomiasis at continental and country level: Update to 2018. PLoS Neglected Trop. Dis. 2020, 14, e0008261 10.1371/journal.pntd.0008261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Priotto G.; Kasparian S.; Mutombo W.; Ngouama D.; Ghorashian S.; Arnold U.; Ghabri S.; Baudin E.; Buard V.; Kazadi-Kyanza S.; Ilunga M.; Mutangala W.; Pohlig G.; Schmid C.; Karunakara U.; Torreele E.; Kande V. Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. Lancet 2009, 374, 56–64. 10.1016/S0140-6736(09)61117-X. [DOI] [PubMed] [Google Scholar]
  10. Checchi F.; Piola P.; Ayikoru H.; Thomas F.; Legros D.; Priotto G. Nifurtimox plus Eflornithine for late-stage sleeping sickness in Uganda: a case series. PLoS Neglected Trop. Dis. 2007, 1, e64 10.1371/journal.pntd.0000064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Priotto G.; Kasparian S.; Ngouama D.; Ghorashian S.; Arnold U.; Ghabri S.; Karunakara U. Nifurtimox-eflornithine combination therapy for second-stage Trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in Congo. Clin. Infect. Dis. 2007, 45, 1435–1442. 10.1086/522982. [DOI] [PubMed] [Google Scholar]
  12. Qu N.; Ignatenko N. A.; Yamauchi P.; Stringer D. E.; Levenson C.; Shannon P.; Perrin S.; Gerner E. W. Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine. Biochem. J. 2003, 375, 465–470. 10.1042/bj20030382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bacchi C. J.; Nathan H. C.; Hutner S. H.; McCann P. P.; Sjoerdsma A. Polyamine metabolism: a potential therapeutic target in trypanosomes. Science 1980, 210, 332–334. 10.1126/science.6775372. [DOI] [PubMed] [Google Scholar]
  14. Carbone P. P.; Douglas J. A.; Thomas J.; Tutsch K.; Pomplun M.; Hamielec M.; Pauk D. Bioavailability study of oral liquid and tablet forms of alpha-difluoromethylornithine. Clin. Cancer Res. 2000, 6, 3850–3854. [PubMed] [Google Scholar]
  15. Jansson-Löfmark R.; Na-Bangchang K.; Björkman S.; Doua F.; Ashton M. Enantiospecific reassessment of the pharmacokinetics and pharmacodynamics of oral eflornithine against late-stage Trypanosoma brucei gambiense sleeping sickness. Antimicrob. Agents Chemother. 2015, 59, 1299–1307. 10.1128/AAC.04101-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Van Nieuwenhove S.; Schechter P. J.; Declercq J.; Boné G.; Burke J.; Sjoerdsma A. Treatment of gambiense sleeping sickness in the Sudan with oral DFMO (DL-α-difluoromethylornithine), an inhibitor of ornithine decarboxylase; first field trial. Trans. R. Soc. Trop. Med. Hyg. 1985, 79, 692–698. 10.1016/0035-9203(85)90195-6. [DOI] [PubMed] [Google Scholar]
  17. Taelman H.; Schechter P. J.; Marcelis L.; Sonnet J.; Kazyumba G.; Dasnoy J.; Haegele K. D.; Sjoerdsma A.; Wery M. Difluoromethylornithine, an effective new treatment of Gambian trypanosomiasis. Results in five patients. Am. J. Med. 1987, 82, 607–614. 10.1016/0002-9343(87)90107-0. [DOI] [PubMed] [Google Scholar]
  18. Doua F.; Boa F. Y.; Schechter P. J.; Miézan T. W.; Diai D.; Sanon S. R.; De Raadt P.; Haegele K. D.; Sjoerdsma A.; Konian K. Treatment of human late stage gambiense trypanosomiasis with alpha-difluoromethylornithine (eflornithine): efficacy and tolerance in 14 cases in Cote d’Ivoire. Am. J. Trop. Med. Hyg. 1987, 37, 525–533. 10.4269/ajtmh.1987.37.525. [DOI] [PubMed] [Google Scholar]
  19. Milord F.; Pépin J.; Ethier L.; Milord F.; Loko L.; Ethier L.; Mpia B. Efficacy and toxicity of eflornithine for treatment of Trypanosoma brucei gambiense sleeping sickness. Lancet 1992, 340, 652–655. 10.1016/0140-6736(92)92180-N. [DOI] [PubMed] [Google Scholar]
  20. Taelman H.; Marcelis L.; Sonnet J.; Kazymba G.; Van den Enden E.; Wery M.; Schechter P. Traitement de la trypanosomiase humaine a Trypanosoma brucei gambiense par l’alpha-difluoromethylornithine. Resultats chez 7 patients. (English: Treatment of human trypanosomiasis caused by Trypanosoma brucei gambiense using alpha-difluoromethylornithine. Results in 7 patients). Bull. Soc. Pathol. Exot. Ses Fil. 1988, 81, 578–588. [PubMed] [Google Scholar]
  21. Creaven P. J.; Pendyala L.; Petrelli N. J. Evaluation of alpha-difluoromethylornithine as a potential chemopreventive agent: tolerance to daily oral administration in humans. Cancer Epidemiol., Biomarkers Prev. 1993, 2, 243–247. [PubMed] [Google Scholar]
  22. Abeloff M. D.; Slavik M.; Luk G. D.; Griffin C. A.; Hermann J.; Blanc O.; Sjoerdsma A.; Baylin S. B. Phase I trial and pharmacokinetic studies of alpha-difluoromethylornithine--an inhibitor of polyamine biosynthesis. J. Clin. Oncol. 1984, 2, 124–130. 10.1200/JCO.1984.2.2.124. [DOI] [PubMed] [Google Scholar]
  23. Na-Bangchang K.; Doua F.; Konsil J.; Hanpitakpong W.; Kamanikom B.; Kuzoe F. The pharmacokinetics of eflornithine (alpha-difluoromethylornithine) in patients with late-stage T.b. gambiense sleeping sickness. Eur. J. Clin. Pharmacol. 2004, 60, 269–278. 10.1007/s00228-004-0759-7. [DOI] [PubMed] [Google Scholar]
  24. Griffin C. A.; Slavik M.; Chien S. C.; Hermann J.; Thompson G.; Blanc O.; Luk G. D.; Baylin S. B.; Abeloff M. D. Phase I trial and pharmacokinetic study of intravenous and oral α-difluoromethylornithine. Invest. New Drugs 1987, 5, 177–186. 10.1007/BF00203544. [DOI] [PubMed] [Google Scholar]
  25. Pepin J.; Guern C.; Milord F.; Schechter P. J. Difluoromethylornithine for arseno-resistant Trypanosoma brucei gambiense sleeping sickness. Lancet 1987, 330, 1431–1433. 10.1016/S0140-6736(87)91131-7. [DOI] [PubMed] [Google Scholar]
  26. Horn Y.; Schechter P. J.; Marton L. J. Phase I-II clinical trial with alpha-difluoromethylornithine - an inhibitor of polyamine biosynthesis. Eur. J. Cancer Clin. Oncol. 1987, 23, 1103–1107. 10.1016/0277-5379(87)90141-6. [DOI] [PubMed] [Google Scholar]
  27. Love R. R.; Carbone P. P.; Verma A. K.; Gilmore D.; Carey P.; Tutsch K. D.; Pomplun M.; Wilding G. Randomized phase I chemoprevention dose-seeking study of alpha-difluoromethylornithine. J. Natl. Cancer Inst. 1993, 85, 732–737. 10.1093/jnci/85.9.732. [DOI] [PubMed] [Google Scholar]
  28. Talpaz M.; Plager C.; Quesada J.; Benjamin R.; Kantarjian H.; Gutterman J. Difluoromethylornithine and leukocyte interferon: a phase I study in cancer patients. Eur. J. Cancer Clin. Oncol. 1986, 22, 685–689. 10.1016/0277-5379(86)90166-5. [DOI] [PubMed] [Google Scholar]
  29. Andersson S.; Allenmark S. G. Preparative chiral chromatographic resolution of enantiomers in drug discovery. J. Biochem. Biophys. Methods 2002, 54, 11–23. 10.1016/S0165-022X(02)00126-4. [DOI] [PubMed] [Google Scholar]
  30. Miller L. Preparative enantioseparations using supercritical fluid chromatography. J. Chromatogr. A 2012, 1250, 250–255. 10.1016/j.chroma.2012.05.025. [DOI] [PubMed] [Google Scholar]
  31. Leek H.; Thunberg L.; Jonson A. C.; Öhlen K.; Klarqvist M. Strategy for large-scale isolation of enantiomers in drug discovery. Drug Discovery Today 2017, 22, 133–139. 10.1016/j.drudis.2016.09.018. [DOI] [PubMed] [Google Scholar]
  32. Leek H.; Andersson S. Preparative Scale Resolution of Enantiomers Enables Accelerated Drug Discovery and Development. Molecules 2017, 22, 158. 10.3390/molecules22010158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Malm M.; Bergqvist Y. Determination of eflornithine enantiomers in plasma, by solid-phase extraction and liquid chromatography with evaporative light-scattering detection. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 846, 98–104. 10.1016/j.jchromb.2006.08.030. [DOI] [PubMed] [Google Scholar]
  34. Hanpitakpong W.; Kamanikom B.; Banmairuroi V.; Na-Bangchang K. High-performance liquid chromatographic method for determination of 2-difluoromethyl-DL-ornithine in plasma and cerebrospinal fluid. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2003, 788, 221–231. 10.1016/S1570-0232(02)00438-5. [DOI] [PubMed] [Google Scholar]
  35. Jansson R.; Malm M.; Roth C.; Ashton M. Enantioselective and nonlinear intestinal absorption of eflornithine in the rat. Antimicrob. Agents Chemother. 2008, 52, 2842–2848. 10.1128/AAC.00050-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yang S.; Peng K.-W.; Wang M. Z. A simple and sensitive assay for eflornithine quantification in rat brain using pre-column derivatization and UPLC-MS/MS detection. Biomed. Chromatogr. 2015, 29, 918–924. 10.1002/bmc.3374. [DOI] [PubMed] [Google Scholar]
  37. Wagner J.; Gaget C.; Heintzelmann B.; Wolf E. Resolution of the enantiomers of various α-substituted ornithine and lysine analogs by high-performance liquid chromatography with chiral eluant and by gas chromatography on Chirasil-Val. Anal. Biochem. 1987, 164, 102–116. 10.1016/0003-2697(87)90374-5. [DOI] [PubMed] [Google Scholar]
  38. Jansson-Löfmark R.; Römsing S.; Albers E.; Ashton M. Determination of eflornithine enantiomers in plasma by precolumn derivatization with o-phthalaldehyde-N-acetyl-L-cysteine and liquid chromatography with UV detection. Biomed. Chromatogr. 2010, 24, 768–773. 10.1002/bmc.1361. [DOI] [PubMed] [Google Scholar]
  39. Smithers J. A precolumn derivatization high-performance liquid chromatographic (HPLC) procedure for the quantitation of difluoromethylornithine in plasma. Pharm. Res. 1988, 05, 684–686. 10.1023/A:1015943408386. [DOI] [PubMed] [Google Scholar]
  40. Huebert N. D.; Schwartz J. J.; Haegele K. D. Analysis of 2-difluoromethyl-DL-ornithine in human plasma, cerebrospinal fluid and urine by cation-exchange high-performance liquid chromatography. J. Chromatogr. A 1997, 762, 293–298. 10.1016/S0021-9673(96)00818-7. [DOI] [PubMed] [Google Scholar]
  41. Grove J.; Fozard J. R.; Mamont P. S. Assay of α-difluoromethylornithine in body fluids and tissues by automatic amino-acid analysis. J. Chromatogr. 1981, 223, 409–416. 10.1016/S0378-4347(00)80114-0. [DOI] [PubMed] [Google Scholar]
  42. Alvarez-Coque M. C. G.; Hernández M. J. M.; Camañas R. M. V.; Fernández C. M. Formation and instability of o-phthalaldehyde derivatives of amino acids. Anal. Biochem. 1989, 178, 1–7. 10.1016/0003-2697(89)90346-1. [DOI] [PubMed] [Google Scholar]
  43. Simons S. S. Jr.; Johnson D. F. Reaction of o-phthalaldehyde and thiols with primary amines: fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles. Anal. Biochem. 1978, 90, 705–725. 10.1016/0003-2697(78)90163-X. [DOI] [PubMed] [Google Scholar]
  44. Yoshikawa K.; Furuno M.; Tanaka N.; Fukusaki E. Fast enantiomeric separation of amino acids using liquid chromatography/mass spectrometry on a chiral crown ether stationary phase. J. Biosci. Bioeng. 2020, 10.1016/j.jbiosc.2020.05.007. [DOI] [PubMed] [Google Scholar]
  45. Miller L.; Yue L. Chiral separation of underivatized amino acids in supercritical fluid chromatography with chiral crown ether derived column. Chirality 2020, 32, 981–989. 10.1002/chir.23204. [DOI] [PubMed] [Google Scholar]
  46. Caldwell J. Do single enantiomers have something special to offer?. Hum. Psychopharmacol. 2001, 16, S67–s71. 10.1002/hup.339. [DOI] [PubMed] [Google Scholar]
  47. Lu H. Stereoselectivity in drug metabolism. Expert Opin. Drug Metab. Toxicol. 2007, 3, 149–158. 10.1517/17425255.3.2.149. [DOI] [PubMed] [Google Scholar]
  48. Äbelö A.; Andersson T. B.; Antonsson M.; Naudot A. K.; Skånberg I.; Weidolf L. Stereoselective metabolism of omeprazole by human cytochrome P450 enzymes. Drug Metab. Dispos. 2000, 28, 966–972. [PubMed] [Google Scholar]
  49. Tybring G.; Böttiger Y.; Widén J.; Bertilsson L. Enantioselective hydroxylation of omeprazole catalyzed by CYP2C19 in Swedish white subjects. Clin. Pharmacol. Ther. 1997, 62, 129–137. 10.1016/S0009-9236(97)90060-6. [DOI] [PubMed] [Google Scholar]
  50. Echizen H.; Manz M.; Eichelbaum M. Electrophysiologic effects of dextro- and levo-verapamil on sinus node and AV node function in humans. J. Cardiovasc. Pharmacol. 1988, 12, 543–546. 10.1097/00005344-198811000-00007. [DOI] [PubMed] [Google Scholar]
  51. Agranat I.; Caner H.; Caldwell J. Putting chirality to work: the strategy of chiral switches. Nat. Rev. Drug Discovery 2002, 1, 753–768. 10.1038/nrd915. [DOI] [PubMed] [Google Scholar]
  52. EMA Investigation of chiral active substances; European Medicines Agency: https://www.ema.europa.eu/en/investigation-chiral-active-substances, 1993, pp 381–391.
  53. FDA’s policy statement for the development of new stereoisomeric drugs. Chirality 1992, 4, 338–340. 10.1002/chir.530040513. [DOI] [PubMed] [Google Scholar]
  54. Johansson C.-C.; Gennemark P.; Artursson P.; Äbelö A.; Ashton M.; Jansson-Löfmark R. Population pharmacokinetic modeling and deconvolution of enantioselective absorption of eflornithine in the rat. J. Pharmacokinet. Pharmacodyn. 2013, 40, 117–128. 10.1007/s10928-012-9293-x. [DOI] [PubMed] [Google Scholar]
  55. Brown A. P.; Morrissey R. L.; Crowell J. A.; Levine B. S. Thirteen-week oral toxicity study of difluoromethylornithine in combination with tamoxifen citrate in female dogs. Cancer Chemother. Pharmacol. 1999, 43, 479–488. 10.1007/s002800050927. [DOI] [PubMed] [Google Scholar]
  56. Kirchner D. L.; Mercieca M. D.; Crowell J. A.; Levine B. S. Developmental toxicity studies of 2-(difluoromethyl)-dl-ornithine (DFMO) in rats and rabbits. Toxicol. Sci. 1999, 50, 127–135. 10.1093/toxsci/50.1.127. [DOI] [PubMed] [Google Scholar]
  57. Loiseau P. M.; Chauffert O.; Czok M. Effect of chronic trypanosomiasis on the bioavailability of alpha-difluoromethylornithine (DFMO) after oral administration: pharmacokinetics study on DFMO plasma levels in infected and noninfected mice using a high-performance liquid chromatography assay. Parasitol. Res. 1997, 83, 386–389. 10.1007/s004360050268. [DOI] [PubMed] [Google Scholar]
  58. Burri C.; Brun R. Eflornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 2003, 90, S49–S52. 10.1007/s00436-002-0766-5. [DOI] [PubMed] [Google Scholar]
  59. Cloete T. T.; Johansson C. C.; N’Da D. D.; Vodnala S. K.; Rottenberg M. E.; Breytenbach J. C.; Ashton M. Mono-, di- and trisubstituted derivatives of eflornithine: synthesis for in vivo delivery of DL-α-difluoromethylornithine in plasma. Arzneimittelforschung 2011, 61, 317–325. 10.1055/s-0031-1296205. [DOI] [PubMed] [Google Scholar]
  60. Helena K. J.; N’Da D. D.; Johansson C. C.; Breytenbach J. C.; Ashton M. Effects of oral administration of synthesized delta-amides of eflornithine in the rat. Arzneimittelforschung 2010, 60, 682–688. 10.1055/s-0031-1296347. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao0c03121_si_001.pdf (212.6KB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES