S-adenosyl methionine (SAME) and valproic acid (VPA) combinations for preventing VPA-induced congenital malformations

Abstract

The present invention relates to a combination of valproic acid (VPA) and S- adenosylmethionine (SAMe) for use in preventing congenital malformations in a fetus of a subject in need of VPA treatment and who is pregnant or intends to become pregnant, by administering the combination to the subject before or during the first trimester of pregnancy, wherein the SAMe and the VPA are at a ratio of about 1:10-1:50 w / w SAMe:VPA in the combination; and corresponding methods of treatment.

Description

[0001] S-ADENOSYL METHIONINE (SAMe) AND VALPROIC ACID (VPA) COMBINATIONS FOR PREVENTING VPA-INDUCED CONGENITAL MALFORMATIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally directed to prevention of congenital malformations induced during pregnancy by valproic acid (VPA). More specifically, the invention relates to early administration of S-adenosyl methionine (SAMe) in addition to Valproic acid (VPA) during pregnancy for prevention of embryonic malformations, especially neural tube defects (NTD) caused by VPA administration.

[0004] BACKGROUND OF THE INVENTION

[0005] Valproic acid (VPA) is a very effective antiepileptic and mood stabilizing drug. It is used for the treatment of convulsive disorder, bipolar disorder, neuralgia and has lately been used in clinical trials for the treatment of several types of cancer (prostatic and breast cancer). However, it is a highly teratogenic drug and therefore its use during pregnancy is contraindicated. If administered in the first trimester of pregnancy, during organogenesis, it may induce a variety of congenital malformations, especially neural tube defects (meningomyelocele), cardiac, skeletal and orofacial malformations. VPA is also a very potent neuroteratogen in humans, causing a high rate of cognitive impairment, attention deficit disorder (ADHD) and autism spectrum disorder (ASD).

[0006] Animal models, especially using mice and rats, have demonstrated similar teratogenic and neuroteratogenic effects mimicking the effects of VPA in pregnant women. Among the most important mechanism of teratogenicity / neurodevelopmental toxicity by VPA are effects on gene expression via inhibition of histone deacetylase, thus promoting DNA demethylation and inducing uncoordinated gene expression. Many attempts were made to reduce the teratogenic and neurotoxic effects of VPA by modifying the chemical structure of VPA while trying to maintain its therapeutic potency, but so far these attempts have been unsuccessful.

[0007] SAMe is a methyl donor for methyltransferases. Of particular importance is its activity as an intermediate in the synthesis of norepinephrine, dopamine, and serotonin, suggesting its potential role in mood regulation. SAMe is widely used as a food additive, and used medically as adjunct treatment for depression and other "epigenetic" diseases. It is not known to cause any teratogenic effects. Since SAMe affects genomic methylation patterns in different and opposing ways than VPA, it has a potential to be used for preventing the adverse effects of VPA. Recent publications appear to support the use of SAMe for preventing autism spectrum disorders (ASD) caused by VP A administered during pregnancy or after birth, but not for preventing congenital malformations caused by VPA administered during the first trimester of pregnancy (Omoy et al. 2019, Neurotoxicology and Teratology 71 :64-74; Weinstein-Fudim et al. 2019, Int. J. Mol. Sci. 20(21): 5278; Weinstein- Fudim et al. 2020, IJMS 21(8), pii: E2834; Omoy et al. 2023, Gender specific neurobehavioral and gene expression changes in a valproic acid (VPA) - induced mouse model of autistic like behavior and correction by S-adenosylmethionine (SAMe). In: Sex, Gender and Epigenetics, Chapter 14: 163-179, Ubeda-Martin et al., 1998, Nutr. Hosp. 13:41-49).

[0008] Accordingly, there is a need for a solution that will allow administration of VPA during pregnancy without resulting in teratogenic and neurotoxic effects, such as a drug that would block the teratogenic and neurotoxic activities of VPA.

[0009] SUMMARY OF INVENTION

[0010] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0011] In some embodiments, there is provided a combination of valproic acid (VPA) and S- adenosylmethionine (SAMe) for use in a method of preventing congenital malformations in a fetus of a subject in need of VPA treatment and who is pregnant or intends to become pregnant, the method including administering the combination to the subject before or during the first trimester of pregnancy, wherein the SAMe and the VPA are at a ratio of about 1 : 10-1 :50 w / w SAMe: VPA in the combination.

[0012] In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :30 w / w SAMe:VPA in the combination.

[0013] In some embodiments, the weight ratio between the administered SAMe and the administered VPA is maintained constant throughout pregnancy of the subject.

[0014] In some embodiments, the number of administrations of the VPA and the SAMe during pregnancy are the same. In some embodiments, the number of daily administrations of the VPA and the SAMe during pregnancy are the same

[0015] In some embodiments, the combination is administered during the pregnancy 3 or fewer times.

[0016] In some embodiments, the VPA and the SAMe are administered together. In some embodiments, the VPA and the SAMe are included in a single composition. In some embodiments, the SAMe is administered no later than one day following each administration of the VPA.

[0017] In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy no later than one day following each administration of the VPA during pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy prior to or at the same time as each administration of the VPA.

[0018] In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy within 2 hours prior to each administration of the VPA.

[0019] In some embodiments, the subject has been diagnosed with a disorder or condition selected from a convulsive disorder, a bipolar disorder, neuralgia, or breast cancer.

[0020] In some embodiments, there is provided a method of preventing congenital malformations in in a fetus of a subject in need of valproic acid (VPA) treatment and who is pregnant or intends to become pregnant, the method including administering to the subject a combination of VPA and S-adenosylmethionine (SAMe) before or during the first trimester of pregnancy, wherein the SAMe and the VPA are at a ratio of about 1 : 10-1 :50 w / w SAMe: VPA in the combination.

[0021] In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :30 w / w SAMe:VPA in the combination.

[0022] In some embodiments, the weight ratio between the administered SAMe and the administered VPA is maintained constant throughout pregnancy of the subject.

[0023] In some embodiments, the number of administrations of the VPA and the SAMe during pregnancy are the same. In some embodiments, the number of daily administrations of the VPA and the SAMe during pregnancy are the same

[0024] In some embodiments, the VPA and the SAMe are administered to the subject during pregnancy only as part of the combination. In some embodiments, the combination is administered during the pregnancy 3 or fewer times.

[0025] In some embodiments, the VPA and the SAMe are administered together. In some embodiments, the VPA and the SAMe are included in a single composition. In some embodiments, the SAMe is administered no later than 1 day following each administration of the VPA.

[0026] In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy no later than one day following each administration of the VPA. In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy prior to or at the same time as each administration of the VPA.

[0027] In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy within 2 hours prior to each administration of the VPA.

[0028] In some embodiments, each administration of the VPA during the pregnancy is preceded by administration of SAMe at a ratio of about 1 : 10 w / w SAMe: VPA.

[0029] In some embodiments, the subject has been diagnosed with a disorder or condition selected from a convulsive disorder (such as epilepsy), a bipolar disorder, neuralgia, or breast cancer.

[0030] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0031] BRIEF DESCRIPTION OF DRAWINGS

[0032] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0033] Fig- 1 shows four 15 day old mouse fetuses. Upper panels: left - saline treatment (normal looking), right - VPA treatment (showing severe exencephaly); lower panels: left - SAMe treatment (normal looking), right - VPA + SAMe treatment (normal looking).

[0034] Figs. 2A-2B show weight and length of fetuses at gestation day 15. Fig. 2A. fetal weight in gram (g). Fig. 2B. Crown-Rump Length (CRL) in centimeter (cm). Treatments (left to right): control (Saline); VPA alone; SAMe alone; VPA + SAMe. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: PO.OOl (***).

[0035] Figs. 3A-3B show % of death (Fig. 3A) and % exencephaly (Fig. 3B) in fetuses at gestation day 15. Treatments (left to right): control (Saline); VPA alone; VPA + SAMe; lyophilized VPA + SAMe mix; SAMe alone. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: P<0.01 (**), P<0.001 (***), P<0.0001 (****).

[0036] Fig. 4 shows malondialdehyde (MDA) levels in brains of fetuses at gestation day 15. Treatments (left to right): control (Saline); VPA alone; VPA + SAMe; SAMe alone. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: PO.OOl (***), P0.0001 (****). TBARS - thiobarbituric acid reactivity species.

[0037] Figs. 5A-5B show catalase activity and gene expression in brains of fetuses at gestation day 15. Fig. 5A. Activity in all samples. Fig. 5B. Gene expression, samples not including exencephaly fetuses. Treatments (left to right): control (Saline); VPA alone; VPA + SAMe; lyophilized VPA + SAMe (only in Fig. 5B); SAMe alone. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: P<0.01 (**).

[0038] Figs. 6A-6B show Glutathione peroxidase (GPx) enzyme activity and gene expression in brains of fetuses at gestation day 15. Fig. 6A. Activity in all samples. Fig. 6B. Gene expression, samples not including exencephaly fetuses. Treatments (left to right): control (Saline); VPA alone; VPA + SAMe; lyophilized VPA + SAMe (only in Fig. 6B); SAMe alone. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: P<0.01 (**).

[0039] Fig- 7 shows NOS1 gene expression in brains of fetuses at gestation day 15 (excluding exencephaly samples). Treatments (left to right): control (Saline); VPA alone; VPA + SAMe; lyophilized VPA + SAMe; SAMe alone. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: P<0.05 (*), P<0.01 (**), P<0.001 (***).

[0040] Fig. 8 shows NOS2 gene expression in brains of fetuses at gestation day 15 (excluding exencephaly samples). Treatments (left to right): control (Saline); VPA alone; VPA + SAMe; lyophilized VPA + SAMe; SAMe alone. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: P<0.05 (*), P<0.01 (**), P<0.001 (***).

[0041] Fig. 9 shows MECP2 gene expression in brains of fetuses at gestation day 15 (excluding exencephaly samples). Treatments (left to right): control (Saline); VPA alone; VPA + SAMe; lyophilized VPA + SAMe; SAMe alone. Asterisks represent Bonferroni’s multiple comparison test adjusted P value: P<0.05 (*).

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0044] In recently published work, the inventors focused on preventing the cognitive and behavioral effects of valproic acid (VPA) administered during pregnancy or shortly thereafter, such as autism spectrum disorder (ASD), and have shown that administration of S-adenosylmethionine (SAMe) late during pregnancy or postnatally in addition to VPA was capable of preventing ASD symptoms and changes in neurophysiological and pathological gene expression caused by VPA when administered alone (Omoy et al. 2019, Neurotoxicology and Teratology 71 :64-74; Weinstein- Fudim et al. 2019, Int. J. Mol. Sei. 20(21): 5278; Weinstein- Fudim et al. 2020, IJMS 21(8), pii: E2834; Omoy et al. 2023, Gender specific neurob ehavi oral and gene expression changes in a valproic acid (VP A) - induced mouse model of autistic like behavior and correction by S- adenosylmethionine (SAMe). In: Sex, Gender and Epigenetics, Chapter 14: 163-179). The administration during pregnancy was done in mice at gestation day 12, which corresponds to postorganogenesis, after which time VPA treatment is known to cause neurobehavioral problems, such as ASD.

[0045] However, VPA has additional, more detrimental, teratogenic effects, including congenital malformations, especially neural tube defects (potentially leading, inter alia, to spina bifida, exencephaly), which most likely result from its early administration during pregnancy.

[0046] The mechanisms of the teratogenic effects of VPA (enhanced oxidative stress in the brain and inhibition of histone deacetylase) are different from those responsible for its beneficial anti convulsive and mood stabilizing effects (blocks voltage gated ion channels and increases GABAergic synaptic activity) and may well also be different from those causing the more severe teratogenic effects.

[0047] Additionally, in an experiment testing co-administration of VPA and SAMe early during pregnancy in rats, SAMe addition showed no protective effects compared to VPA alone, as manifested by fertility index and the amount of resorptions and (Ubeda-Martin et al., 1998, Nutr. Hosp. 13:41-49). A later publication by the same group (Ubeda et al., 2000, European Journal of Anatomy 4(l):23-33) concluded that coadministration of VPA and SAMe did not prevent malformations, stating that skeletal malformations were significantly elevated as compared to other groups. The administration schedule of Ubeda involved administration of 300 / mg / kg / day VPA at gestation days 8, 9, 10, and 10 mg / kg / day SAMe at gestation days 1-10, which translates to a ratio of SAMe:VPA of 1 :30 on the days both are administered. The authors further state on page 31 : “However, the administration of elevated SAMe concentrations could cause a metabolic disruption in the methylation cycle that would enhance the effect of VPA resulting in alterations to foetal morphogenesis”. It is further noted that testing of congenital malformations is difficult since the mothers tend to eat the malformed offspring. Since, as noted above, the Ubeda schedule was not successful, it is possible that a different administration schedule might lead to a better outcome.

[0048] In the present application, the inventors surprisingly show successful prevention of VPA neural tube malformations induced during the first trimester of pregnancy, during organogenesis by using 300 mg / kg / day VPA and 30mg / kg / day SAMe, which translates to a ratio of SAMe to VPA of 1 : 10 w / w, which, as opposed to Ubeda, is kept constant for all administrations. By keeping constant it is meant that the administration of VPA and SAMe according to the present invention during pregnancy requires that both agents are administered each time within several hours or a day of one another, unlike Ubeda, in which SAMe was administered during the first 7 days of gestation before the first VPA administration. The effect was examined on day 15 of pregnancy rather than at full term, since by then the fetuses are severely malformed and usually already dead and resorbed.

[0049] In Example 1 described below, pregnant Institute of Cancer Research (ICR) mice were injected with 300 mg / Kg of VPA during days 8 and 9 of gestation, with or without 30mg / kg SAMe administered on the same gestational days, one hour prior to VPA injection. During days 8 and 9 of mouse gestation VPA administration is known to induce a variety of congenital malformations, especially 20%-30% of exencephaly as well as cardiac, orofacial and other malformations. As shown in Table 1 for 15 day old mouse fetuses, treatment with SAMe reduced the resorption rate to control levels (Fig. 3A) and abolished exencephaly (Fig. 3B). Additionally, a lyophilized mixture of VPA and SAMe (10: 1 w / w) was also shown to prevent exencephaly, reduce resorption, and normalize fetal weight to control levels.

[0050] As further shown in Examples 2 and 3, pre-administration of SAMe was also able to prevent or reduce effects of VPA related to oxidative stress (Figs. 4-6), nitrosative stress (Figs. 7-8), and chromatin acetylation (Fig. 9). These effects, which are alleviated by SAMe administration, are related to mechanisms involved in VPA-induced teratogenesis and neural tube defects.

[0051] The combination and the method described herein are therefore intended for a subject who is treated by VPA or needs VPA treatment (e.g., due to epileptic seizures, and as further explained below) and is pregnant or may become pregnant. Since the VPA may cause congenital malformations (such as neural tube defects) in a prospective fetus of the subject, the combination and method disclosed herein provide administration of SAMe together with or before each administration of the VPA in order to prevent the congenital malformations.

[0052] Combinations of VPA and SAMe for administration

[0053] In some embodiments, there is provided a combination of VPA and SAMe for use in a method of preventing congenital malformations in a fetus of a subject in need of VPA treatment and who is pregnant or intends to become pregnant, the method including administering the combination to the subject before or during the first trimester of pregnancy, wherein the SAMe and the VPA are at a ratio of about 1 : 10-1 :50 w / w SAMe:VPA in the combination.

[0054] In some embodiments, the subject is a human subject.

[0055] It is appreciated that the meaning of the herein disclosed combination (as further described below) is that the weight ratio between the administered SAMe and the administered VPA is maintained constant throughout pregnancy of the subject. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 :20 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 : 15 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :20-1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10, 1 : 15, 1 :20, 1 :25, or 1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 15 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :20 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :25 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :30 w / w SAMe:VPA in the combination.

[0056] In some embodiments, each administration of the VPA during pregnancy is accompanied by an administration of the SAMe. In some embodiments, each administration of the SAMe during pregnancy is accompanied by an administration of the VPA. In some embodiments, the number of administrations of the VPA and the SAMe during pregnancy are the same. In some embodiments, the number of daily administrations of the VPA and the SAMe during pregnancy are the same.

[0057] The term “accompanied”, as used herein, means that for each administration of the VPA (or the SAMe), the other one is also administered, according to weight ratios and timing definitions further defined herein.

[0058] In some embodiments, the combination is administered to the subject during pregnancy fewer than 5, 4, 3 or 2 times. In some embodiments, the combination is administered to the subject during pregnancy fewer than 3 times. In some embodiments, the combination is administered to the subject during pregnancy once. In some embodiments, the combination is administered to the subject during pregnancy at least twice. In some embodiments, the combination is administered to the subject during pregnancy twice. In some embodiments, the combination is administered to the subject during pregnancy three times.

[0059] The term “preventing congenital malformations”, as used herein, relates to the fact that VPA is known to cause congenital malformations, especially neural tube defects potentially leading, inter alia, to spina bifida and / or exencephaly, when administered early during pregnancy at the time of neural tube formation. Preventing congenital malformations means that the amount of such congenital malformations is significantly reduced by administering the combination of the invention, such that VPA-induced congenital malformations are largely or completely prevented. In some embodiments, the congenital malformations are neural tube defects (NTDs). In some embodiments, the congenital malformations are spina bifida and / or exencephaly. In some embodiments, the congenital malformations are spina bifida. In some embodiments, the congenital malformations are exencephaly.

[0060] The term “intends to become pregnant”, as used herein, means that the subject may become pregnant, e.g., the subject is at the fertility age and is not using contraception, or the subject is actively trying to become pregnant. In such cases, VPA administration may cause a risk of congenital malformations in a prospective fetus unless the subject is also administered with SAMe, as defined herein.

[0061] The term “before” with respect to pregnancy of the subject relates to a subject who is not pregnant but may become pregnant (a subject who intends to become pregnant, as defined above), regardless of whether or not the subject finally becomes pregnant.

[0062] The reference to “an embryo” or “a fetus” relates to a prospective embryo or fetus that develops once the subject becomes pregnant.

[0063] In some embodiments, the subject is pregnant. In some embodiments, the subject is not pregnant.

[0064] In some embodiments, the combination includes the VPA and the SAMe in a single composition. In some embodiments, the composition is a lyophilized composition. A. In some embodiments, the combination includes at least two compositions, one including VPA and another including SAMe. In some embodiments, the composition or compositions are pharmaceutical compositions.

[0065] In some embodiments, the VPA and the SAMe are administered to the subject together. The term “together”, or simultaneously, means at about the same time, or that one is administered immediately after the other.

[0066] In some embodiments, the VPA and the SAMe are administered to the subject separately. In some embodiments, the VPA and the SAMe are administered at any order. In some embodiments, the VPA is administered at the same time or after administration of the SAMe. In some embodiments, the VPA and the SAMe are administered to the subject separately, with no more than 1 day, 10 hours, 5 hours, 2 hours, or 1 hour between the two administrations.

[0067] It is believed that to prevent the defects caused by VPA, the SAMe should be administered prior to the formation of the defects, i.e. before, or as soon as possible after, administration of the VPA.

[0068] In some embodiments, the SAMe is administered no later than one day following each administration of the VPA. In some embodiments, the SAMe is administered prior to each administration of the VPA. In some embodiments, the SAMe is administered between about 1 day before and about one day after each administration of the VPA.

[0069] In some embodiments, the subject is pregnant and the VPA is administered from the first day of the pregnancy. In some embodiments, the subject is pregnant and human and the first dose of VPA during pregnancy is administered on the first, second, or third month of the pregnancy.

[0070] Accordingly, in some embodiments, the subject is pregnant and the SAMe is administered during pregnancy no later than one day following each administration of the VPA. In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy prior to each administration of the VPA. In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy between about 1 day before and about one day after each administration of the VPA. In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy between about 1 hour before and about 1, 2, 3, 4, 5, 10, 15, 20, or 24 hours after each administration of the VPA.

[0071] In some embodiments, the subject is pregnant and the SAMe is administered within 2 hours prior to each administration of the VPA during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered within 1 hour prior to each administration of the VPA during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered about 1 hour prior to each administration of the VPA during the pregnancy.

[0072] In some embodiments, the VPA is continued to be administered during pregnancy after the first administration of the VPA. In some embodiments, the SAMe is continued to be administered during pregnancy after the first administration of the SAMe.

[0073] In some embodiments, the combination is further administered during the second and / or the third trimester of the pregnancy. In some embodiments, the combination is further administered to the newborn after birth.

[0074] In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe: VPA ratio of about 1 : 10-1 :30 w / w within 1 hour prior to each administration of the VPA during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe:VPA ratio of about 1 : 10-1 :20 w / w within 1 hour prior to each administration of the VPA during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe:VPA ratio of about 1 : 10-1 : 15 w / w within 1 hour prior to each administration of the VPA during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe:VPA ratio of about 1 :20-1 :30 w / w within 1 hour prior to each administration of the VPA during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe: VPA ratio of about 1 : 10 w / w within 1 hour prior to each administration of the VP A during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe:VPA ratio of about 1 : 15 w / w within 1 hour prior to each administration of the VP A during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe: VP A ratio of about 1 :20 w / w within 1 hour prior to each administration of the VP A during the pregnancy. In some embodiments, the subject is pregnant and the SAMe is administered at a SAMe:VPA ratio of about 1 :30 w / w within 1 hour prior to each administration of the VPA during the pregnancy.

[0075] It is appreciated that the term “within” with reference to time, e.g., within 1 hour or within 2 hours, also encompasses administration at the same time, or together.

[0076] To prevent neural tube defects, SAMe should be administered to the subject during the time of neural tube formation, and it is not necessary to administer SAMe prior to that time.

[0077] Accordingly, in some embodiments, the SAMe is administered to the subject only during neural tube formation and / or immediately preceding neural tube formation. In some embodiments, the combination is administered to the subject only during neural tube formation and / or immediately preceding neural tube formation.

[0078] In some embodiments, the subject is a human subject, and the SAMe or the combination is not administered to the subject during the first 2 weeks of the pregnancy. In some embodiments, the subject is a human subject, and the SAMe or the combination is not administered to the subject during the first 3 weeks of the pregnancy.

[0079] The VPA used may be any VPA suitable for administration, for example, sodium valproate, or valproic acid, and including slow / extended release preparations.

[0080] In some embodiments, the SAMe is a stabilized SAMe. In some embodiments, the SAMe is not a stabilized SAMe. In some embodiments, the SAMe is selected from SAMe tosylate disulfate, SAMe butanedi sulfonate, SAMe ademetionine disulfate tosylate, and SAMe P-toluenesulfonate. In some embodiments, the SAMe is selected from SAMe tosylate disulfate, SAMe butanedi sulfonate, and SAMe ademetionine disulfate tosylate. In some embodiments, the SAMe is not SAMe P-toluenesulfonate.

[0081] In some embodiments, the composition or at least one of the at least two compositions further include at least one agent which is typically administered in the third trimester of pregnancy, such as folic acid, prenatal vitamins (e.g. vitamin B12 complex), iron supplements, progesterone, etc.

[0082] VPA is used clinically as an anticonvulsant for seizure disorders, a mood stabilizer for bipolar disorder, and for the prevention of migraines. It works primarily by increasing the levels of gamma-aminobutyric acid (GABA) in the brain and modulating ion channels to stabilize neuronal activity.

[0083] Accordingly, in some embodiments, the subject in need of VP A treatment has been diagnosed with a disorder or condition indicated for VPA treatment. In some embodiments, the subject in need of VPA treatment has been diagnosed with a disorder or condition selected from a convulsive disorder, a bipolar disorder, neuralgia, or breast cancer. In some embodiments, the convulsive disorder is selected from epilepsy, tonic-clonic seizures, absence seizures, myoclonic seizures, atonic seizures, Lennox-Gastaut syndrome, Dravet syndrome, febrile seizures, infantile spasms, status epilepticus, and focal seizures.

[0084] Methods of administering combinations of VPA and SAMe

[0085] In some embodiments, there is provided a method of preventing congenital malformations in a fetus of a subject in need of VPA treatment and who is pregnant or intends to become pregnant, the method including administering to the subject before or during the first trimester of pregnancy a combination of valproic acid (VPA) and S-adenosylmethionine (SAMe), wherein the SAMe and the VPA are at a ratio of about 1 : 10-1 :50 w / w SAMe:VPA in the combination.

[0086] Definitions and embodiments mentioned above and which may be relevant to the embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0087] In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 :20 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10-1 : 15 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :20-1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10, 1 : 15, 1 :20, 1 :25, or 1 :30 w / w SAMe:VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 10 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 : 15 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :20 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :25 w / w SAMe: VPA in the combination. In some embodiments, the SAMe and the VPA are at a ratio of about 1 :30 w / w SAMe:VPA in the combination. In some embodiments, the weight ratio between the administered SAMe and the administered VP A is maintained constant throughout pregnancy of the subject.

[0088] In some embodiments, the SAMe and the VPA are administered to the subject during pregnancy only as part of the combination.

[0089] In some embodiments, the number of administrations of the VPA and the SAMe during pregnancy are the same. In some embodiments, the number of daily administrations of the VPA and the SAMe during pregnancy are the same.

[0090] In some embodiments, the combination is administered to the subject during pregnancy fewer than 5, 4, 3 or 2 times. In some embodiments, the combination is administered to the subject during pregnancy fewer than 3 times. In some embodiments, the combination is administered to the subject during pregnancy once. In some embodiments, the combination is administered to the subject during pregnancy twice.

[0091] In some embodiments, the subject is pregnant. In some embodiments, the subject is not pregnant.

[0092] In some embodiments, the VPA and the SAMe are administered together. In some embodiments, the VPA and the SAMe are included in a single composition. In some embodiments, the composition is a lyophilized composition. In some embodiments, the combination includes at least two compositions, one including the VPA and another including the SAMe.

[0093] In some embodiments, the VPA and the SAMe are administered separately.

[0094] In some embodiments, the SAMe is administered no later than 1 day following each administration of the VPA. In some embodiments, the SAMe is administered prior to administration of the VPA.

[0095] In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy prior to each administration of the VPA.

[0096] In some embodiments, the subject is pregnant and the SAMe is administered during pregnancy within 2 hours prior to each administration of the VPA.

[0097] In some embodiments, the subject has been diagnosed with a disorder or condition selected from a convulsive disorder (such as epilepsy), a bipolar disorder, neuralgia, or breast cancer.

[0098] In some embodiments, the VPA is administered orally, parenterally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or rectally. In some embodiments, the VPA is administered orally. In some embodiments, the VPA is administered subcutaneously. In some embodiments, the VPA is administered intramuscularly. In some embodiments, the VPA is administered intraperitoneally.

[0099] In some embodiments, the SAMe is administered orally, parenterally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or rectally. In some embodiments, the SAMe is administered orally. In some embodiments, the SAMe is administered subcutaneously. In some embodiments, the SAMe is administered intramuscularly. In some embodiments, the SAMe is administered intraperitoneally.

[0100] In some embodiments, the composition including both VPA and SAMe is administered orally, parenterally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or rectally. In some embodiments, the composition including both VPA and SAMe is administered orally. In some embodiments, the composition including both VPA and SAMe is administered subcutaneously. In some embodiments, the composition including both VPA and SAMe is administered intramuscularly. In some embodiments, the composition including both VPA and SAMe is administered intraperitoneally.

[0101] In some embodiments, the composition or the at least two compositions are pharmaceutical compositions, further including a pharmaceutically acceptable carrier. In some embodiments, the composition or the at least two compositions include the VPA and / or the SAMe encapsulated in a dosage form for oral administration. Nonlimiting examples for suitable dosage forms for oral administration include tablets, capsules, liquid, or sprinkle capsules.

[0102] Pharmaceutical compositions for use in accordance with the present invention may be formulated in any conventional manner using one or more physiologically or pharmaceutically acceptable carriers or excipients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition, not being deleterious to the recipient thereof, and not significantly interfering with the activity of the compound of the invention, or of any other active ingredient in the pharmaceutical composition.

[0103] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition may include a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

[0104] The following exemplification of carriers, modes of administration, dosage forms, etc., are listed as known possibilities from which the carriers, modes of administration, dosage forms, etc., may be selected for use with the present invention. Those of ordinary skill in the art will understand, however, that any given formulation and mode of administration selected should first be tested to determine that it achieves the desired results.

[0105] The term “treating” or “treatment”, as used herein, refers to means of obtaining a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease and / or symptoms attributed to the disease. The term includes inhibiting the disease, i.e. arresting its development; or ameliorating the disease, i.e. causing regression of the disease, e.g., by eliminating or ameliorating its symptoms.

[0106] The term “preventing”, as used herein, refers to causing a condition or symptoms thereof not to appear in the subject, or delaying the onset of such condition or symptoms, such that they do not appear at the time they are expected to appear based on similar cases, or causing the condition or symptoms to appear at a diminished level.

[0107] The determination of the doses of the active ingredient to be used for human use is based on commonly used practices in the art, and will be finally determined by physicians in clinical trials. An expected approximate equivalent dose for administration to a human can be calculated based on the in vivo experimental evidence disclosed herein below, using known formulas (e.g. Reagan- Show et al. (2007) Dose translation from animal to human studies revisited. The FASEB Journal 22:659-661). According to this paradigm, the adult human equivalent dose (mg / kg body weight) equals a dose given to a mouse (mg / kg body weight) multiplied with 0.081.

[0108] In some embodiments, the dosage of VP A is about 10-60 mg / kg / day. In some embodiments, the VPA is administered 2 or 3 times daily. In some embodiments, the dosage of SAMe is about 1-6 mg / kg / day.

[0109] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0110] The term "a" and "an" refers to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0111] The term "about", when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0112] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow. The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0113] EXAMPLES

[0114] Example 1: SAMe prevents the teratogenic effects of VPA

[0115] The ability of S-adenosyl methionine (SAMe) to prevent neural tube defects induced by Valproic acid (VPA) was tested.

[0116] Institute of Cancer Research (ICR) mice were intraperitoneally injected during days 8 and 9 of gestation with two injections of 300 mg / Kg each of VPA sodium salt (sodium valproate, CAS No: 1069-66-5, CatNo: P4543, Sigma Aldrich, Merck, IL) in saline solution, exposing the embryos to treatment on these days of pregnancy. For control, pregnant mice were injected with saline instead of VPA. These days were chosen because VPA administration during this phase of organogenesis is known to induce in mice a variety of congenital malformations, especially 15%- 25% of exencephaly, that is easily observed.

[0117] To examine the possible preventive effect of VPA-induced teratogenicity by SAMe, 30 mg / Kg SAMe (Ademetionine, CAS No. 29908-03-0, CAT No. 1003035, MuseChem, Pennsylvania, USA) were administered intraperitoneally one hour before each VPA injection. For control, saline was administered instead of SAMe.

[0118] On day 15 of the pregnancy, fetuses were evaluated for exencephaly, fetal resorptions (i.e. fetal death) and fetal growth. The results are summarized in Table 1. Brain samples from treated fetuses were also frozen at -80°C for additional testing.

[0119] Additionally, the above experiment was repeated, but instead of separate administration of VPA and SAMe, a lyophilized powder of VPA and SAMe (VPA / SAMe powder) at a ratio of 10 / 1 (300 mg / Kg of VPA and 30mg / Kg SAMe) was administered by intraperitoneal injection to pregnant mice on days 8 and 9 of pregnancy.

[0120] Table 1: Effects of VPA, SAMe and their combination on embryonic development

[0121] * Significantly lower than other groups As seen from Table 1, the levels of resorptions and exencephaly in the VPA + SAMe treated mice were both similar to the control, indicating that administration of the two agents together prevented the congenital abnormalities, specifically exencephaly caused by VPA administered alone. Fig- 1 shows representative fetuses from the four treatments, showing normal looking fetuses for all treatments, except for the VPA treatment (upper right panel), which presents a fetus with exencephaly. Additionally, as also shown in Figs. 2A-2B, the growth retardation caused by VPA was reversed by the addition of SAMe, as seen from the weight (Fig. 2A) and length (Fig. 2B) of the fetuses. As further shown in Table 1 and Figs. 3A-3B, the mixture of VPA and SAMe significantly reduced the VPA-induced death (Fig. 3A) and exencephaly (Fig. 3B) and normalized fetal weight and resorptions, demonstrating the same effects as the separate administration of these substances.

[0122] Example 2: SAMe prevents oxidative stress effects of VPA

[0123] For the oxidative stress assays described below, frozen brain samples of treated fetuses were lysed in IM PBS buffer then homogenized, centrifuged at 14,000g for 10 minutes at 4°C and the protein concentration in the supernatant was quantified using the BCA protein assay kit (CAT #23227, Thermo Scientifica USA). The supernatant was used in all assays of the oxidative stress markers. Absorbance for different assays was obtained using plate reader (TECAN infinite 200 Pro, Austria).

[0124] To assess oxidative stress-induced damage, Malondialdehyde (MDA) levels were measured. MDA is a small organic compound formed as a breakdown product when polyunsaturated fatty acids (PUFAs) in cell membranes undergo lipid peroxidation due to reactive oxygen species (ROS). MDA is widely used as a biomarker for oxidative stress and lipid peroxidation in both plants and animals. MDA levels were measured as follows: the lipid peroxidation product of MDA, which is generated in embryos by free radical injury, was measured by thiobarbituric acid reactivity using spectrophotometric colorimetry by adopting the method described in literatures (Sachett et al., 2020, “Quantification of Thiobarbituric Acid Reactive Species (TBARS) Optimized for Zebrafish Brain Tissue.” protocols. io https: / / dx.doi.org / 10.17504 / protocols.io.bjp8kmrw). MDA concentration was calculated by a calibration curve standardization using Malondialdehyde tetrabutylammonium salt (Sigma-Aldrich CAS #100683543), Trichloroacetic acid (TCA) (Sigma- Aldrich; CAS #76039), 2Thiobarbituric acid (TBA) (Sigma-Aldrich; CAS #504176) and Ethanol (Merck Millipore Catalog #100983). The results are expressed as nmol per pg of protein in the embryo. As shown in Figs. 4A-4B, MDA levels are increased by VPA, indicating stress-induced cellular damage, and normalized by SAMe. To further evaluate oxidative stress, catalase (CAT) activity was measured by photometric determination of catalase activity performed using the potassium permanganate titration method with significant modification (Goldblith and Proctor, Photometric determination of catalase activity, J. Biol. Chem 1950, 18(2) 705-709). Briefly, supernatants were mixed with H2O2 in 1.5ml tubes and left to react for three minute. After three minutes the reaction is stopped by addition of sulfuric acid. With a pipette, the content of each tube is transferred to 96 well plate in triplicate. Excess potassium permanganate is added and within one minute the absorbance is read at 480 nm. Catalase is a key antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into water and oxygen. Since H2O2 is a reactive oxygen species (ROS), catalase is critical for protecting cells from oxidative stress. As shown in Fig. 5A, catalase activity is increased by VPA and normalized by SAMe. Gene expression was also tested, as explained in the next section, and the results shown in Fig. 5B also show an increase of catalase expression by VPA, normalized by SAMe.

[0125] Yet another oxidative stress marker is glutathione peroxidase (GPx), a selenium-dependent antioxidant enzyme. GPx reduces hydrogen peroxide (H2O2) and organic hydroperoxides (ROOH) to water (H2O) or corresponding alcohols, using reduced glutathione (GSH) as a cofactor. GPx thus prevents lipid peroxidation and protects cells against oxidative stress. GPX activity (nmol NADPH / min / ml) was assayed with a Cellular Glutathione Peroxidase assay kit (Sigma-Aldrich Catalog #MAK437). The kit protocol for tissue samples was followed strictly. As can be seen from Fig. 6A, while VPA increased GPx activity, SAMe normalized it. Gene expression was also tested, as explained in the next section, and the results shown in Fig. 6B show an increase of GPx expression by VPA, normalized by SAMe.

[0126] Example 3: Effects of VPA and SAMe on gene expression

[0127] Brain samples were retrieved from -80°c and kept on ice. Using a Dounce homogenizer and pestle, the tissues were homogenized for RNA extraction. Total RNA extraction mini kit (Geneaid CAT #RT D050) was used for the extraction and purification of RNA following the manufactures protocol. The concentration and purity of the extracted RNA was confirmed using NanoDrop (DS- 11 series, DeNovix) and RNA integrity validated on gel. The RNA samples were reverse transcribed to cDNA (Quantabio cDNA synthesis kit Lot #66250414).

[0128] For gene expression determination, the cDNA was subjected to quantitative RT-PCR (Azure Biosystem CIELO) using 2* SYBR green qPCR Master Mix (Quantabio Fastmix Lot #66272685). The thermal profile was 95°C for 2 min and 40 cycles of 95°C for 10 s and 60°C for 30 sec. Relative expression level of the genes was quantified by the 2-AACt method with Hprt being used as endogenous control genes hence, expression level for target genes was normalized by the Ct value of Hprt. Primers used are listed in Table 2.

[0129] Table 2: Primers for gene expression studies

[0130] Further, to test the effect of VPA on nitrosative stress, and whether the addition of SAMe counters that effect, expression of nitrous oxide synthase 1 (NOS1) and nitrous oxide synthase 2 (NOS2) genes was tested in the brain of day 15 fetuses as described above. As can be seen in Figs. 7 (NOS1) and 8 (NOS2), gene expression was increased by VPA and decreased by the addition of SAMe. The decrease of NOS 1 and NOS2 was even below the levels in the saline treated controls.

[0131] MECP2 is an epigenetic regulator that binds methylated DNA and is critical for neuronal maturation, synaptic plasticity, and brain development. VPA is a known histone deacetylase (HDAC) inhibitor, causing chromatic hyperacetylation and DNA demethylation and elevating MECP2 expression. On the other hand, SAMe induces DNA methylation, thereby decreasing gene activity, which should counter the effect of VPA. MECP2 gene expression was tested as described above in brains of fetuses at gestation day 15. As can be seen from Fig. 9, VPA increased the expression of MECP2, however, the addition of SAMe significantly decreased the MECP2 gene expression in relation to VPA. Decreased MECP2 gene expression was also observed in the brains of the exencephalic fetuses in relation to all other groups (data not shown).

[0132] Additionally, VPA increased the expression of IGF1, IGF2 and VEGF which were significantly decreased (to normal levels) following proadministration of SAMe (data not shown).

[0133] Example 4: optimized VPA to SAMe ratio

[0134] ICR mice are intraperitoneally injected during days 8 and 9 of gestation with two injections of 300 mg / Kg of VPA sodium salt each (to a total of 600 mg / kg) in saline solution. For control, pregnant mice are injected with saline instead of VP A. To test for preventive effect on VPA- induced teratogenicity by SAMe, 10, 20, or 30 mg / Kg SAMe are administered intraperitoneally one hour before each VPA injection. For control, saline is administered instead of SAMe. Alternatively, mice are treated with a lyophilized powder of VPA and SAMe at a SAMe: VPA ratio of 1 : 10, 1 : 15, or 1 :30 w / w.

[0135] On day 15 of the pregnancy, fetuses are evaluated for exencephaly, fetal resorptions (i.e., fetal death) and fetal growth, as described in Example 1. Additionally, fetuses are evaluated for effects on additional parameters described in Examples 2 and 3, such as testing for levels of MDA, and activity of catalase and GPx, as well as gene expression levels of SOD1, SOD2, NOS1, NOS2, and MECP2. It is expected that the lower doses of SAMe will also be effective, as shown above for a 1 : 10 ratio.

Claims

1. CLAIMSWhat is claimed is:

1. A combination of valproic acid (VP A) and S-adenosylmethionine (SAMe) for use in a method of preventing congenital malformations in a fetus of a subject in need of VP A treatment and who is pregnant or intends to become pregnant, the method comprising administering the combination to the subject before or during the first trimester of pregnancy, wherein the SAMe and the VPA are at a ratio of about 1 : 10-1 :50 w / w SAMe:VPA in the combination.

2. The combination of claim 1, wherein the SAMe and the VPA at a ratio of about 1 : 10-1 :30 w / w SAMe:VPA.

3. The combination of claim 2, wherein the SAMe and the VPA at a ratio of about 1 : 10 w / w SAMe:VPA.

4. The combination of any one of claims 1-3, wherein the weight ratio between the administered SAMe and the administered VPA is maintained constant throughout pregnancy of the subject.

5. The combination of any one of claims 1-4, wherein the number of administrations of the VPA and the SAMe during pregnancy are the same.

6. The combination of any one of claims 1-5, wherein the number of daily administrations of the VPA and the SAMe during pregnancy are the same.

7. The combination of any one of claims 1-6, wherein the combination is administered during the pregnancy 3 or fewer times.

8. The combination of any one of claims 1-7, wherein the VPA and the SAMe are administered together.

9. The combination of claim 8, wherein the VPA and the SAMe are comprised in a single composition.

10. The combination of any one of claims 1-9, wherein the subject is pregnant and the SAMe is administered during pregnancy prior to or at the same time as each administration of the VPA.

11. The combination of any one of claims 1-10, wherein the subject is pregnant and the SAMe is administered during pregnancy within 2 hours prior to each administration of the VPA.

12. The combination of any one of claims 1-11, wherein the subject has been diagnosed with a disorder or condition selected from a convulsive disorder, a bipolar disorder, neuralgia, or breast cancer.

13. A method of preventing congenital malformations in a fetus of a subject in need of valproic acid (VP A) treatment and who is pregnant or intends to become pregnant, the method comprising administering to the subject a combination of VPA and S-adenosylmethionine (SAMe) before or during the first trimester of pregnancy, wherein the SAMe and the VPA are at a ratio of about 1 : 10-1 :50 w / w SAMe:VPA in the combination.

14. The method of claim 13, wherein the SAMe and the VPA are at a ratio of about 1 : 10-1 :30 w / w SAMe:VPA in the combination.

15. The method of claim 14, wherein the SAMe and the VPA are at a ratio of about 1 : 10 w / w SAMe:VPA in the combination.

16. The combination of any one of claims 13-15, wherein the weight ratio between the administered SAMe and the administered VPA is maintained constant throughout pregnancy of the subject.

17. The combination of any one of claims 13-16, wherein the number of administrations of the VPA and the SAMe during pregnancy are the same.

18. The combination of any one of claims 13-17, wherein the number of daily administrations of the VPA and the SAMe during pregnancy are the same.

19. The method of any one of claims 13-18, wherein the combination is administered during the pregnancy 3 or fewer times.

20. The method of any one of claims 13-19, wherein the VPA and the SAMe are administered together.

21. The method of claim 20, wherein the VPA and the SAMe are comprised in a single composition.

22. The method of any one of claims 13-21, wherein the subject is pregnant and the SAMe is administered during pregnancy prior to or at the same time as each administration of the VPA.

23. The method of any one of claims 13-22, wherein the subject is pregnant and the SAMe is administered during pregnancy within 2 hours prior to each administration of the VPA.

24. The method of any one of claims 13-23, wherein the subject has been diagnosed with a disorder or condition selected from a convulsive disorder (such as epilepsy), a bipolar disorder, neuralgia, or breast cancer.

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