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 taking four other medications concurrently, 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.

[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.

Eflunomine hydrochloride monohydrate is the hydrochloride and hydrate of efornithine, a trypanolytic drug. It is a hydrochloride and hydrate containing efornithine. Eflunomine hydrochloride is the hydrochloride form of efornithine, a difluoromethylated ornithine compound with antitumor activity. Eflunomine irreversibly inhibits ornithine decarboxylase (an enzyme required for polyamine biosynthesis), thereby inhibiting the formation and proliferation of tumor cells. Polyamines participate in nucleosome oligomerization and DNA conformation, thereby creating a chromatin environment that stimulates tumor transformation of cells. (NCI04) Ornithine decarboxylase inhibitor, ornithine decarboxylase is the rate-limiting enzyme in the polyamine biosynthesis pathway. See also: Eflunomine (with active moiety). Drug Indications Treatment of facial hirsutism in women. Eflunomine is the only new molecular drug approved in the past 50 years for the treatment of human trypanosomiasis. It is primarily used as a backup medication for Gambian trypanosomiasis cases resistant to melarsone. The most common dosage regimen for treating Gambian trypanosomiasis sleeping sickness is 100 mg/kg body weight administered intravenously every 6 hours for 14 days (150 mg/kg body weight for children). Due to the high ornithine decarboxylase turnover rate of the trypanosome Trypanosoma brucei rhodesiense, the efficacy of efornithine is limited. Adverse reactions are common during efornithine treatment, characterized similarly to other cytotoxic drugs used to treat cancer. The incidence and severity of adverse reactions increase with treatment duration and deterioration of the patient's general condition. Generally, adverse reactions to efornithine are reversible after treatment. These adverse reactions include seizures (7%), gastrointestinal symptoms such as nausea, vomiting, and diarrhea (10%-39%), myelotoxicity leading to anemia, leukopenia, and thrombocytopenia (25%-50%), hearing impairment (5% in cancer patients), and hair loss (5%-10%). The drug inhibits embryonic development in mice, rats, and rabbits, but its secretion in breast milk is unknown. The mean half-life is approximately 3-4 hours, and the volume of distribution is approximately 0.35 L/kg. Renal clearance is approximately 2 mL/min/kg (intravenous injection), representing over 80% of the drug clearance. The bioavailability of an oral dose of 10 mg/kg is estimated at 54%. One of the key determinants of treatment success appears to be the cerebrospinal fluid drug concentration achieved during treatment; studies have shown that concentrations above 50 μmol/L are necessary for sustained parasite clearance. Because its mechanism of action is to inhibit rather than kill trypanosomes, its onset of action is relatively slow. [1] Eflunomine is a specific, irreversible inhibitor of ornithine decarboxylase, which slows hair growth by inhibiting this enzyme in hair follicles. In women with facial hirsutism (Hypertrichosis), the transdermal absorption rate was less than 1% when 15% efnomine cream was applied twice daily to a 50 cm² area of shaved skin below the chin. In clinical studies of women with facial hirsutism, 15% efnomine cream was demonstrated superior to placebo in reducing hair growth using both objective and subjective methods, with effects observed after 2 to 8 weeks of treatment. After 24 weeks of treatment, 58% of patients in the efnomine group and 34% in the placebo group showed at least improvement in their facial hirsutism symptoms (for ease of analysis, all patients not assessed at week 24 were considered to have worsened or shown no improvement). Furthermore, 32% of patients were considered to have achieved treatment success (at least significant improvement), compared to only 8% in the placebo group. Hair growth returned to pre-treatment levels within 8 weeks of discontinuation of treatment. The study assessed the effects of the treatment on six aspects of patients’ health using a self-assessment questionnaire. The results showed that the average overall discomfort and distress of patients in the efornithine group was reduced by 33% compared with the placebo group (15% in the placebo group). Adverse events mainly affected the skin. Only burning/tingling/numbing sensations were significantly more common in the efornithine group than in the placebo group. [2]

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Background: Facial hirsutism is a cosmetic problem for women and can lead to severe anxiety and low self-esteem. Efluornithine cream is indicated for the treatment of facial hirsutism. However, even with long-term and high-frequency use, its efficacy and overall patient satisfaction remain limited and there is room for improvement. Objective: This study aimed to test the effects of microneedling on the in vitro skin penetration and in vivo efficacy of efornithine cream in a mouse model. Materials and Methods: In vitro penetration of efornithine was studied using a Franz diffusion cell. In vivo efficacy was studied by monitoring hair regeneration in the skin on the lower back of mice after applying efornithine cream to the microneedling pretreated area. Histological examination of the skin and hair follicles in the treatment area was also performed. Results and Discussion: When efornithine cream was applied to the skin area of mice pretreated with microneedles, its activity in inhibiting hair growth was significantly enhanced. This is likely because the micropores formed by the microneedles facilitated the penetration of efornithine into the skin, which was also confirmed by in vitro penetration experiments. Immunohistochemical data showed that when efornithine cream was applied to the skin area pretreated with microneedles, cell proliferation in the skin and hair follicles was also significantly inhibited. Conclusion: Combining microneedling therapy with topical efornithine therapy is a potentially feasible method to enhance the ability of efornithine to inhibit hair growth. Keywords: cell proliferation; inhibiting hair growth; microneedles; skin penetration; excess hair growth. [3] This study investigated the time effect of the polyamine synthesis inhibitor efornithine (α-difluoromethylornithine) on the response of the aortic ring vessels of hypertensive rats with aortic constriction to potassium chloride, norepinephrine, sodium nitroprusside and acetylcholine. Hypertension caused by aortic coarctation reduced the contractile response of the aortic ring to potassium chloride and norepinephrine, increased sensitivity to norepinephrine (reduced EC50 value), and attenuated the diastolic response to acetylcholine after 14 days of hypertension. In rats with hypertension caused by aortic coarctation treated with efornithine, the contractile strength to potassium chloride and norepinephrine and the diastolic response to acetylcholine returned to normal after 14 days of hypertension. The responses to sodium nitroprusside were similar in all groups at all time points. The excessive response to norepinephrine caused by aortic coarctation was not affected by efornithine. These studies suggest that long-term administration of efornithine can restore vascular function to normal in cases of significantly elevated blood pressure. This restoration of function is associated with the regression of structural abnormalities mediated by efornithine that are usually associated with hypertension due to pressure overload. [4]

C6H15CLF2N2O3

236.6447

236.074

C, 30.45; H, 6.39; Cl, 14.98; F, 16.06; N, 11.84; O, 20.28

96020-91-6

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

441361

White to off-white solid powder

1.293g/cm3

347ºC at 760mmHg

>210ºC (dec.)

163.7ºC

1.91

5

7

5

14

166

0

Cl.O=C(C(C(F)F)(CCCN)N)O.O

FJPAMFNRCFEGSD-UHFFFAOYSA-N

InChI=1S/C6H12F2N2O2.ClH.H2O/c7-4(8)6(10,5(11)12)2-1-3-9/h4H,1-3,9-10H2,(H,11,12)1H1H2

2,5-diamino-2-(difluoromethyl)pentanoic acid hydrochloride hydrate

Ornidyl; CPP-1X; RMI71782; Eflornithine hydrochloride hydrate; Eflornithine hydrochloride monohydrate; Vaniqa; Eflornithine HCl; dfmo; MDL 71,782 A; Eflornithine HCl hydrate; RMI-71782; RMI 71782; DL-Ornithine hydrochloride;

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 (~352.14 mM)

Solubility in Formulation 1: 100 mg/mL (422.58 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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