Ornithine decarboxylase

Eflornithine is a specific, irreversible inhibitor of the enzyme ornithine decarboxylase which is thought to slow hair growth by inhibiting this enzyme in hair follicles[2].

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In vitro permeation study of eflornithine was performed using Franz diffusion cell. The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles.[3]

The only novel drug approved in the last fifty years for the treatment of human African trypanosomiasis is eflornithine. It is mostly used as a stand-by medication for Trypanosoma brucei gambiense infections that do not respond to melarsoprol [1]. When it came to decreasing the growth of facial hair in participants with excess hair, eflornithine 15% cream outperformed a placebo. Following a 24-week course of treatment, face hirsutism improved at least somewhat in 58% of eflornithine patients and 34% of placebo subjects [2]. When eflornithine cream was administered to mouse skin areas that had been microneedled beforehand, the hair growth inhibitory activity of eflornithine was markedly increased [3]. After 14 days of hypertension, eflornithine treatment of constrictive hypertensive rats led to the normalization of KCI and norepinephrine contractile strength as well as the relaxing of acetylcholine [4].

In in vitro studies using Salmonella and two strains of Saccharomyces, eflornithine did not induce mutagenic changes [1].

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In vitro permeation of eflornithine hydrochloride through mouse skin[3]
In vitro permeation assay using Franz diffusion cell apparatus was completed as previously described (Kumar et al. 2012; Kumar et al. 2011; Naguib, Kumar, & Cui 2014) using the lower dorsal skin of C57BL/6 mice. Hair was trimmed using an electric clipper 24 h before the collection of the skin. Skin was harvested, wrapped in aluminum foil, and stored at −20°C for a maximum period of one month and used whenever needed. Freezing of the skin at −20°C (without a cryo-protectant) is commonly applied in literature, and such skin samples have been used frequently for permeability studies (Stahl, Wohlert, & Kietzmann 2012). Dennerlein et al. showed that freezing and storing of freshly excised human skin for up to 30 days at −20°C does not affect the skin permeability (Dennerlein et al. 2013). Other researchers showed that when human skin was wrapped in aluminum foil and stored at −26°C, the skin retained its barrier properties for up to 6 months (Badran, Kuntsche, & Fahr 2009). After the fat layer was removed, the skin was mounted onto the Franz diffusion cells with dorsal side facing upward. The receiver compartment contained 5 ml of water and was maintained at 37°C with a Haake SC 100 Water Circulator (ThermoScientific, Wellington, NH). The hair-trimmed skin was treated with a Dermaroller® microneedle roller as previously described before it was mounted onto the Franz diffusion cells (Kumar et al. 2011; Naguib, Kumar, & Cui 2014). The skin sample was placed onto the flat surface of a balance, and the microneedle roller was rolled in four perpendicular directions over the skin surface, 5 times each for a total of 20 times, with an applying pressure of 350–400 g, which was constantly measured using the balance while the roller was rolled. The diffusion area of the skin was 0.64 cm2. The donor compartment was loaded with 4 mg of eflornithine hydrochloride in 500 μl water and covered with parafilm to prevent evaporation. After 0, 1, 3, 6, 8, and 24 h, samples (150 μl) were withdrawn from the receiver compartment and immediately replenished with fresh medium. The samples were analyzed using HPLC following a method described previously with modifications (Saravanan et al. 2009). Chromatographic analysis was carried out with an Agilent 1260 Infinity HPLC station equipped with ZORBAX Eclipse Plus C18 (5 μm, 4.6 × 150 mm) column using a acetonitrile-buffer mixture (70%:30%, v/v) as the mobile phase. The buffer was prepared by dissolving 0.68 g of potassium phosphate monobasic in 1 l of water. The flow rate was 0.8 ml/min. The detector wavelength was 210 nm.

Skin tissues were fixed with a buffered formalin (10%) solution for 24 h, washed with 0.1 M of sodium phosphate buffer (pH 7.4), dehydrated in graded ethanol, embedded in paraffin, and sectioned vertically. The sections were stained using hematoxylin-eosin (H&E) or an antibody against 5-bromo-2′-deoxyuridine (BrdU) in the Histology and Tissue Processing facility in the Dell Pediatric Research Institute at the University of Texas at Austin. Mice were injected intraperitoneally with BrdU in phosphate buffered saline (PBS, pH 7.4, 10 mM) at the dose of 100 μg/g body weight, 30 min prior to euthanasia. All skin sections were examined under an Olympus BX53 microscope [3].

In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically[3].
Female C57BL/6 mice (8–10 weeks old) were are ideal for examining the physiological actions during different hair cycle phases due to the occurrence of naturally synchronized hair cycles with cyclic pigmentation (Slominski, Paus, & Costantino 1991). Each experimental group was composed of 3–4 mice. Hair in the lower dorsal skin of anesthetized mice was either trimmed using an electric clipper, plucked using GiGi® Honee warm wax as previously described (Xiao et al. 2012), or chemically removed using Nair® lotion. The skin area where the hair was removed was then treated with the eflornithine hydrochloride 13.9% cream (~50 mg per mouse per treatment) using a spatula two times a day in an interval of at least 8 h for a maximum period of 36 days. A group of mice whose hair in the application site was trimmed using a clipper were also treated with the microneedle roller every time before the application of eflornithine cream as previously described (Kumar et al. 2012). Briefly, mice were placed onto the flat surface of a balance, and the microneedle roller was rolled over the marked skin surface, 10 times parallel to mouse length, with an applying pressure of 350–400 g as indicated on the balance. In control groups, the hair in mouse dorsal skin was removed by trimming, plucking, or chemical depilation with Nair®, but the area was not treated with the eflornithine cream. The hair re-growth was evaluated by taking digital photographs of the mouse skin areas for a maximum period of 36 days after the first application of the eflornithine cream. On the last day of the study, animals were euthanized, and skin samples were collected from the treated areas for immunohistochemical studies.[3]

Absorption, Distribution and Excretion
Following oral administration of efornithine, the peak plasma concentration (Cmax) of efornithine is reached at 3.5 hours post-administration (Tmax). Food (high-fat and high-calorie) does not affect the Cmax and AUC (area under the concentration-time curve) of efornithine. Crushing the tablet and adding it to a standard pudding mixture has no effect on the exposure to efornithine (Cmax and AUC6h). Under clinical use conditions, for women with excess facial hair, the mean transdermal absorption of the 13.9% (w/w) efornithine cream formulation, after single or multiple administrations, is less than 1% of the radioactive dose. Clinical use conditions include shaving within 2 hours prior to administration of the radiolabeled dose, as well as other forms of facial hair removal such as shaving, plucking, or tweezing. Steady state is reached within four days with twice-daily administration. Under clinical use conditions, in 10 women with excess facial hair (n=10), applying 0.5 g of cream twice daily (total dose 1.0 g/day; equivalent to 139 mg of anhydrous efornithine hydrochloride) resulted in steady-state Cmax, Ctrough, and AUC12hr of approximately 10 ng/mL, 5 ng/mL, and 92 ng hr/mL, respectively, expressed as anhydrous free base of efornithine hydrochloride. Under steady-state conditions, with twice-daily application of 0.5 g of cream (total dose 1.0 g/day), the dose-normalized peak concentration (Cmax) and daily systemic exposure (AUC) of efornithine are expected to be approximately 100-fold and 60-fold lower, respectively, than with a once-daily oral dose of 370 mg. This compound is not metabolized and is primarily excreted unchanged in the urine. The volume of distribution (Vz/F) of efornithine is 24.3 L. The clearance (CL/F) of efornithine is 5.3 L/h. Under clinical use conditions, in female patients with facial hirsutism, the mean transdermal absorption of efornithine after a single or multiple doses of the 13.9% (w/w) cream formulation was less than 1% of the radioactive dose. Clinical use conditions included shaving within 2 hours prior to application of the radiolabeled drug. Apart from other methods of facial hair removal such as cutting, plucking, or tweezing, under clinical use conditions, in women (n=10) with excess facial hair, after twice-daily application of 0.5 g of the cream (total dose 1.0 g/day; equivalent to 139 mg of anhydrous efornithine hydrochloride), the steady-state Cmax, Ctrough, and AUC12hr, expressed as free base of anhydrous efornithine hydrochloride, were approximately 10 ng/mL, 5 ng/mL, and 92 ng/mL, respectively.
At steady state, compared to 370 mg daily, the dose-normalized peak concentration (Cmax) and daily systemic exposure (AUC) of efflunitine were estimated to be reduced by approximately 100-fold and 60-fold, respectively, by twice-daily application of 0.5 g of cream (total dose 1.0 g/day). Oral administration once daily.
Eflunitine is not metabolized and is excreted unchanged in the urine.
For more complete data on the absorption, distribution, and excretion of efflunitine (8 metabolites), please visit the HSDB record page.

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Metabolism/Metabolites
This compound is not metabolized and is primarily excreted unchanged in the urine.

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Biological Half-Life
The terminal plasma elimination half-life of efflunitine is 3.5 hours, and the apparent steady-state plasma half-life is approximately 8 hours.
The apparent steady-state plasma t1/2 of efflunitine is approximately 8 hours.

Effects During Pregnancy and Lactation
◉ Overview of Lactation Use
Mothers received daily intravenous infusions of 400 mg/kg efflunitine for 7 days without any serious adverse effects on breastfed infants. Topical efflunitine is poorly absorbed and therefore unlikely to enter the infant's bloodstream, and is unlikely to cause any adverse effects on breastfed infants.
◉ Effects on Breastfed Infants
In the Democratic Republic of Congo, a cohort study of 33 infants followed hospitalized mothers taking nifurulimus who were breastfeeding (feeding extent not specified). 30 mothers completed 30 doses of oral nifurulimus (15 mg/kg/day), and all mothers received 14 intravenous infusions of efflunitine (400 mg/kg/day) for 7 days for the treatment of human African trypanosomiasis (sleeping sickness). On average, breastfeeding mothers were concurrently taking four other medications, including amoxicillin, ciprofloxacin, metronidazole, sulfamethoxazole/trimethoprim, aspirin, and diclofenac (1 case each); hydrocortisone, promethazine, and quinine (2 cases each); levamisole (6 cases); sulfadoxine-pyrimethamine (8 cases); aminopyrine (13 cases); acetaminophen (16 cases); and mebendazole (17 cases). No serious adverse events were reported in any of the breastfed infants.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.
Protein binding
Effornithine does not specifically bind to human plasma proteins.

[1]. Eflornithine for the treatment of human African trypanosomiasis. Parasitol Res. 2003 Jun;90 Supp 1:S49-52.

[2]. Topical eflornithine. Am J Clin Dermatol. 2001;2(3):197-201; discussion 202.

[3]. A method to improve the efficacy of topical eflornithine hydrochloride cream. Drug Deliv. 2016 Jun;23(5):1495-501.

[4]. Eflornithine alters changes in vascular responsiveness associated with coarctation hypertension. Clin Exp Hypertens. 1997 Apr;19(3):297-312.

Eflornithine is a fluoroamino acid with a difluoromethyl group substituted at the 2-position of ornithine. It is a trypanolytic agent. Eflornithine is a fluoroamino acid and also an α-amino acid whose function is related to ornithine. Eflornithine is an irreversible ornithine decarboxylase inhibitor, originally developed for the treatment of African trypanosomiasis in humans. Further research has shown that ornithine decarboxylase is also associated with other diseases, such as facial hirsutism and cancer, especially when ornithine decarboxylase is highly upregulated in tumor cells. Furthermore, ornithine decarboxylase can be activated by c-myc or interact with ras, both of which are well-known oncogenes; therefore, there is growing interest in targeting ornithine decarboxylase as a potential cancer treatment. In 1960 and 2000, the FDA approved flunominosine under the brand names ORNIDYL and VANIQUA for the treatment of African trypanosomiasis and hirsutism, respectively, but its use has since been discontinued. Subsequently, on December 14, 2023, the FDA reapproved efflunitine under the brand name IWILFIN as an oral maintenance therapy to reduce the risk of relapse in high-risk neuroblastoma patients in adults and children who have at least a partial response to prior multi-drug, multimodal therapy (including anti-GD2 immunotherapy). This approval was based on positive results from a multicenter, single-arm, externally controlled study in children with high-risk neuroblastoma, which observed a 52% reduction in relapse risk and a 68% reduction in mortality risk. efflunitine is an antiprotozoal drug and a decarboxylase inhibitor. Its mechanism of action is as a decarboxylase inhibitor. efflunitine is a difluoromethylated ornithine compound with antitumor activity. It irreversibly inhibits ornithine decarboxylase (an enzyme required for polyamine biosynthesis), thereby inhibiting tumor cell formation and proliferation. Polyamines are involved in nucleosome oligomerization and DNA conformation, creating a chromatin environment that stimulates tumor transformation. This drug has been shown to induce apoptosis in leiomyoma cells. (NCI04)
Ornithine decarboxylase inhibitor. Ornithine decarboxylase is the rate-limiting enzyme in the polyamine biosynthesis pathway.
See also: Eflunomine hydrochloride (salt form).
Drug Indications
Eflunomine is indicated for reducing the risk of relapse in adult and pediatric patients with high-risk neuroblastoma (HRNB) who have previously received multi-drug combination therapy, including anti-GD2 immunotherapy, and have achieved at least partial remission. It was previously used to treat hirsutism in women and trypanosomiasis in Africa, but this use has been discontinued.
FDA Label
Treatment of hirsutism in women's face.
Mechanism of Action
Eflunomine is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate-limiting enzyme in the polyamine biosynthesis pathway and a transcriptional target of MYCN. Polyamines are involved in the differentiation and proliferation of mammalian cells and are crucial for tumor transformation. Currently, no studies have investigated the inhibition of ornithine decarboxylase (ODC) in human skin after topical application of efornithine. However, literature reports that oral efornithine inhibits ODC activity in the skin. It is speculated that topical application of efornithine hydrochloride may irreversibly inhibit skin ODC activity. This enzyme is essential for polyamine synthesis. Animal experimental data indicate that inhibition of ornithine decarboxylase inhibits cell division and synthesis functions, thereby affecting hair growth rate. VANIQA has shown a hair growth-delaying effect in both non-clinical and clinical studies. Eflunomine hydrochloride (α-difluoromethylornithine) has an inhibitory effect on hair growth. The mechanism by which topical efornithine hydrochloride inhibits hair growth is not fully elucidated. Multiple studies of oral efornithine hydrochloride suggest that the drug may inhibit ornithine decarboxylase (ODC), an enzyme that catalyzes the biosynthesis of intracellular polyamines, which are essential for cell division and differentiation. Limited animal experimental data suggest that this inhibition of cell division and differentiation may affect hair growth rate. The manufacturer of topical efornithine hydrochloride states that there are currently no published studies on the inhibitory effect of topical efornithine hydrochloride on human ODC.

C6H12F2N2O2

182.16848

182.087

C, 39.56; H, 6.64; F, 20.86; N, 15.38; O, 17.57

70052-12-9

Eflornithine hydrochloride hydrate;96020-91-6;L-Eflornithine monohydrochloride;69955-42-6;Eflornithine hydrochloride;68278-23-9;L-Eflornithine;66640-93-5; 70052-12-9; 70050-55-4 (R-isomer)

3009

White to light yellow solid

1.293g/cm3

347ºC at 760 mmHg

163.7ºC

1.173

3

6

5

12

166

0

NC(CCCN)(C(F)F)C(O)=O

VLCYCQAOQCDTCN-UHFFFAOYSA-N

InChI=1S/C6H12F2N2O2/c7-4(8)6(10,5(11)12)2-1-3-9/h4H,1-3,9-10H2,(H,11,12)

2,5-diamino-2-(difluoromethyl)pentanoic acid.

CPP-1X; DFMO; MDL 71782; MDL-71782; EFLORNITHINE; 70052-12-9; dfmo; Difluoromethylornithine; Ornidyl; 2-(Difluoromethyl)ornithine; 2,5-diamino-2-(difluoromethyl)pentanoic acid; Eflornithinum; MDL71782; RMI71782; RMI-71782; RMI 71782; Difluoromethylornithine Eflornithine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~83.33 mg/mL (~457.43 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)