Polymeric compositions of monomethyl fumarate and their use in treating relapsing remitting multiple sclerosis and psoriasis

Abstract

The present invention comprises compounds of formula (I): where R.sup.1 may be PEG or other polymeric moieties. For example, R.sup.1 may be repeating PEG units —(CH.sub.2—CH.sub.2—O).sub.n—, wherein n=1-455. R.sup.1 may also be other polymeric moieties of varying sizes and structures, for example, R.sup.1 may be poly(glycolide), poly(lactic acid), poly(lactide), poly(caprolactone), poly(lactide-co-caprolactone), poly(lactide-co-glycolide), or poly(lactic acid)-butanol. One or more embodiments of the invention may also relate to injectable pharmaceutical compositions comprising polymer conjugated monomethyl fumarate, and methods for treating relapsing-remitting multiple sclerosis and psoriasis. ##STR00001##

Claims

1. An injectable dosage form comprising an injectable pharmaceutical composition comprising a compound of formula (I): ##STR00006## wherein R1 represents a polymeric moiety selected from the group consisting of polyethylene glycol (PEG), poly(glycolide), poly(lactic acid), poly(lactide), poly(caprolactone), poly(lactide-co-caprolactone), poly(lactide-co-glycolide), and poly(lactic acid)-butanol, wherein the composition is adapted for administration to a patient parenterally.

2. The composition of claim 1 wherein R1 represents PEG having repeating units —(CH2-CH2-O)n-, wherein n=1-455.

3. The composition of claim 2 wherein the PEG moiety is PEG-400 having n=8-10.

4. The composition of claim 2 wherein the PEG moiety is PEG-1000 having n=21-25.

5. The composition of claim 2 wherein the PEG moiety is PEG-2000 having n=43-48.

6. The composition of claim of 2 wherein the PEG moiety is PEG-3000 having n=65-71.

7. The composition of claim 2 wherein the PEG moiety is PEG-5000, having n=112-117.

8. The composition of claim 2 wherein the PEG moiety has a molecular weight of approximately between 950 and 1050.

9. The composition of claim 2 wherein the compound of formula (I) has the 400 MHz proton NMR spectrum of FIG. 2A.

10. The composition of claim 2 wherein the compound of formula (I) has the 400 MHz proton NMR spectrum of FIG. 2B.

11. The composition of claim 2 wherein the compound of formula (I) has the following 400 MHz proton NMR spectrum in deuterated chloroform (CDCl3): δ 3.33 (s, 3H, OCH3 of mPEG), 3.44-3.75 (m, backbone OCH2CH2O of mPEG), 3.79 (s, 3H, OCH3 of MMF), 4.35 (m, 2H, OCH2 of mPEG adjacent to MMF), 6.87 (m, 2H, —CH═CH— of MMF).

12. The composition of claim 2 wherein the compound of formula (I) has the following 400 MHz proton NMR spectrum in deuterated chloroform (CDCl3): δ 6.87 (s, 2H, —CH═CH— of MMF), 4.35 (m, 2H, OCH2 of mPEG adjacent to MMF), 3.81 (s, 3H, OCH3 of MMF) 3.43-3.76 (m, backbone OCH2CH2O of mPEG), 3.36 (s, 3H, OCH3 of PEG).

13. The composition of claim 2 wherein the compound of formula (I) has a molecular weight of 2192.8 g/mol.

14. The composition of claim 1 wherein the polymeric moiety comprises a straight chain.

15. The composition of claim 1 wherein the polymeric moiety comprises a branched chain.

16. The composition of claim 1 wherein the polymeric moiety comprises a substituted chain.

17. The composition of claim 1 wherein the polymeric moiety comprises an unsubstituted chain.

18. The composition of claim 1 wherein the polymeric moiety is globular.

19. A method for treating relapsing-remitting multiple sclerosis or psoriasis comprising administering to a patient parenterally an injectable pharmaceutical composition comprising a compound of formula (I) ##STR00007## wherein R1 represents a polymeric moiety selected from the group consisting of polyethylene glycol (PEG), poly(glycolide), poly(lactic acid), poly(lactide), poly(caprolactone), poly(lactide-co-caprolactone), poly(lactide-co-glycolide), and poly(lactic acid)-butanol.

20. The method of claim 19, wherein the polymeric moiety is polyethylene glycol (PEG).

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1A shows an HPLC chromatogram of MMF-PEG1000 monitored with a 210 nm UV detector (retention time 3.53 min). The retention time of MMF is 2.11 min.

(2) FIG. 1B shows an HPLC chromatogram of MMF-PEG 2000 monitored with a 210 nm UV detector (retention time 3.10 min). The retention time of MMF is 2.11 min and the peak at 2.66 min is from mobile phase.

(3) FIG. 2A shows the proton NMR spectra of MMF-PEG1000 conjugate. Deuterated chloroform (CDCl3) was used as solvent.

(4) FIG. 2B shows the proton NMR spectra of MMF-PEG2000 conjugate. Deuterated chloroform (CDCl3) was used as solvent.

(5) FIG. 3A. MALDI TOF mass spectrum of PEG2000 (m/z=2080.9).

(6) FIG. 3B. MALDI TOF mass spectrum of MMF-PEG2000 conjugate (m/z=2192.8).

(7) FIG. 4 shows the release kinetics of MMF-PEG conjugates (1 kDa and 2 kDa) in PBS.

(8) FIG. 5 shows the release kinetics of MMF-PEG conjugates (1 kDa and 2 kDa) in 80% human plasma.

(9) FIG. 6 shows a plot of MMF pharmacokinetics, showing plasma concentration vs. time following subcutaneous administration of MMF-PEG2000 conjugate in mice.

DETAILED DESCRIPTION

(10) In an embodiment, the present invention provides a monomethyl fumarate (MMF) derivative useful for the treatment of relapsing-remitting multiple sclerosis (RRMS) that alleviates the side effects of oral dimethyl fumarate (DMF), and provides stable injectable compositions that have superior pharmacokinetic properties. In an embodiment, this invention provides an MMF-polymer conjugate according to formula (I)

(11) ##STR00004##
where R.sup.1 may comprise polyethylene glycol (PEG) or other polymeric moieties. In an embodiment, R.sup.1 may comprise repeating PEG units —(CH.sub.2—CH.sub.2—O).sub.n—, wherein n=1-455. R.sup.1 may also comprise other polymeric moieties of varying sizes and structures, for example, poly(glycolide), poly(lactic acid), poly(lactide), poly(caprolactone), poly(lactide-co-caprolactone), poly(lactide-co-glycolide), or poly(lactic acid)-butanol.

(12) PEG is supplied in various molecular weight grades, for example, PEG-1000 has a molecular weight of about 1000, which may be stated as between approximately 950 and 1050. In an embodiment, this invention uses PEG with a molecular weight of 400 (n=8-10), 1000 (n=21-25), 2000 (n=43-48), 3000 (n=65-71), or 5000 (n=112-117).

(13) In an embodiment, other polymers may be useful in this invention, including poly(glycolide), poly(lactic acid), poly(lactide), poly(caprolactone), poly(lactide-co-caprolactone), poly(lactide-co-glycolide), or poly(lactic acid)-butanol.

(14) In an embodiment, the polymer of this invention may be a straight chain, a branched chain, a substituted chain, and unsubstituted chain, or globular.

(15) Advantages of pharmaceutical compositions comprising PEGylated, or other polymer conjugated MMF derivatives disclosed herein include, for example: increased bioavailability at lower doses; predictable drug-release profile over a defined period of time following each injection; better patient compliance; ease of application; improved systemic availability by avoidance of first-pass metabolism; reduced dosing frequency (i.e., fewer injections) without compromising the effectiveness of the treatment; decreased incidence of side effects; and overall cost reduction of medical care.

(16) Polyethylene Glycol (PEG) and PEGylation

(17) Polyethylene glycol (PEG) can be linked via an ester linkage to drug molecules to exert desirable effects on a molecule that may be immunoreactive or rapidly metabolized. PEG is non-toxic, non-immunogenic, non-antigenic, highly soluble in water and FDA approved. PEG-drug conjugates have several advantages: a prolonged residence in body, a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity. The covalent attachment of PEG to a drug can increase its hydrodynamic size (size in solution), which prolongs its circulatory time by reducing renal clearance (Knop et al., 2010, Veronese et al., 2005 and Harris et al., 2003).

(18) The linkage of PEG to drug molecules is called “PEGylation.” Several PEGylated drugs have been approved, including peptide and non-peptide drugs. A few examples of PEGylated drugs include: Pegaptanib (brand name MACUGEN®); Antihemophilic Factor (Recombinant), PEGylated, (brand name ADYNOVATE®), and peginterferon beta-1a (brand name PLEGRIDY®)).

(19) Preparation of Monomethyl Fumarate-PEG Conjugates

(20) A synthetic route to monomethyl fumarate-polyethylene glycol (MMF-PEG) conjugates is shown in eq. 2.

(21) ##STR00005##

(22) The PEG conjugates were produced by a coupling reaction between PEGs and MMF in presence of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and dimethyl amino pyridine (DMAP) (see experimental section). Following purification, the products were confirmed by NMR and mass spectroscopic analysis.

(23) The purity of the final MMF-PEG conjugates was confirmed by reverse-phase HPLC monitored at 210 and 260 nm, where no such significant amounts of starting material as well as impurities present (FIGS. 1A and 1B). The percentage purity of the MMF-PEG conjugates are 97.7% (1.0 kDa PEG, retention time 3.53 min) and 96.5% (2.0 kDa PEG, retention time 3.10 min) at 210 nm. The retention time of MMF is 2.11 min and the absence of MMF peak at 260 nm confirm the formation of the conjugates in both cases.

(24) In the .sup.1H NMR of MMF-PEG1000 conjugate shown in FIG. 2A, the presence of a characteristic peak at 4.35 ppm for OCH.sub.2 group of PEG confirmed the formation of ester bond between MMF and PEG. Additionally, other peaks related to both chemical entities are present such as singlet at 3.79 ppm for methoxy group of MMF, singlet at 3.33 ppm for methoxy group of PEG, multiplets at 3.44-3.75 for the backbone OCH.sub.2 groups of PEG, and a singlet at 6.87 ppm for two protons (CH═CH) of MMF confirmed the formation of the conjugate. In a similar manner, the structure of MMF-PEG2000 conjugate was established shown in FIG. 2B.

(25) The molecular weight of both MMF-conjugates was evaluated using MALDI TOF mass spectroscopy. The molecular weight of the MMF-PEG1000 conjugate is 1223.5 g/mol when we used PEG1000 as starting materials. The average molecular weight of PEG1000 is 1039.4 g/mol which implies that there are 22-23 OCH.sub.2—CH.sub.2O repeating units present in the PEG chain. Based on molecular weights of PEG1000 and corresponding conjugate, it was determined that one MMF molecule attached to the PEG. This finding is in good agreement with the NMR analysis of the conjugate. In a similar way the structure of MMF-PEG2000 conjugate (m/z=2192.8 g/mol) was established. A representative MALDI TOF spectra of PEG2000 and MMF-PEG2000 conjugate are shown in FIGS. 3A and 3B respectively. In both proton NMR and in MALDI TOF mass cases, it was established that approximately one molecule of MMF reacted with the corresponding PEG molecules.

(26) In-Vitro Drug Release Study of the MMF-PEG Conjugates

(27) In vitro MMF release characteristics of the inventive MMF-PEG conjugates were studied to investigate their stability in physiologically relevant solutions such as PBS (pH 7.4) and in human plasma. 80% Human plasma was used to in the study simulate the biological conditions of intravenous injection.

(28) Drug Release Study in PBS: A drug release study of the MMF-PEG conjugates was performed in 0.1 M phosphate buffer (pH 7.4) at 37° C. A concentration of 3 mg/mL of the conjugate was placed in a water bath and the temperature of the bath was maintained at 37° C. with constant mixing. Samples were collected at appropriate time points and lyophilized using liquid nitrogen. The MMF-PEG conjugates along with released MMF were extracted from the lyophilized powder using acetonitrile and the samples were analyzed in reverse-phase HPLC. The peaks of MMF-PEG conjugates and MMF were monitored at 210 and 260 nm respectively. Since the MMF also further degrades, peak area of MMF-PEG conjugate was used for the analysis.

(29) The results of the study of MMF-PEG 1000 and 2000 conjugates in PBS is shown in FIG. 4. In PBS, both the conjugates released the drug in a steady manner and the release pattern is nearly zero order. Approximately 90% of the drug payload was released in 7 days in both cases with no initial burst. The drug from PEG1000 conjugate is releasing much slower than PEG2000 conjugate. In HPLC the released MMF was monitored at 260 nm. After 4 days, the ester bond of MMF of other end started degrading resulted to a di-acid form of monomethylfumarate.

(30) Drug Release Study in Plasma: Another MMF release study from the MMF-PEGylated conjugates (3 mg/mL) was performed using human pooled plasma diluted to 80% with 0.1 M PBS in a water bath (Dual-action shaker; Polyscience) at 37° C., collecting 200 μL aliquots of the sample at appropriate intervals. The protein was precipitated using 2 M trichloroacetic acid, cold acetonitrile was added, centrifuged, filtered, and stored at −80° C. for HPLC analysis. Different solvents may be used to extract the drug conjugates as well as the released drug from the plasma solution. Each sample was analyzed using HPLC, and the drug release was calculated using a calibration graph. DMF was used as the reference drug for this study.

(31) The results of the plasma study are shown in FIG. 5. In human plasma, the extent of release was relatively higher for both MMF-PEG conjugates, compared to PBS at the same time point. This type of release pattern is expected because of the presence of enzymes (e.g. esterase) which can specifically cleave the ester bond much faster than in case of PBS. Both conjugates are very similar, with the PEG1000 conjugate showing slightly faster release during the initial time points. There are burst release of MMF in case of plasma in initial point (31% at 0 h in case of 2 kDa and 39% at 0 h in case of 1 kDa). Similar to PBS release, the drug started degrading after the 4th day, the peak for the degradation product was very prominent compared to the drug peak. The peak area corresponds to the degraded product was included in the calculation. Based on the plasma release profile, a slow release of the drug from the conjugates in vivo is expected.

(32) Pharmacokinetics in Mice

(33) A pilot pharmacokinetics study was done to determine the route of administration and best possible corresponding dose to get the therapeutic level of MMF in blood. MMF conjugates (500 μg of MMF equivalent) were injected intravenous (IV) and subcutaneous (SC) in 5-6 weeks old BALB-NeuT mice and MMF concentration was determined with HPLC up to day 7. The MMF concentration were increased from 1 h to 24 h and roughly 10% of the MMF was detected in blood plasma at 24 h in both cases. The drug concentration steadily decreased until day 7 in both cases, and MMF concentration in the plasma was in the range of 11-18 μg/mL on day 7 in SC and IV routes of administration. The MMF concentrations in the plasma are higher in case of PEG2000 as compared to PEG1000 in SC and IV route. The peak plasma concentrations of MMF were higher following SC route compared to IV route of injection at all time points.

(34) Since the MMF concentration in blood is reported for Tecfidera (Tecfidera label), our aim was to match the same drug concentration in the blood in mice. The average C.sub.max value of MMF in blood was 2.74 μg/mL (360 mg single dose) and 2.15 μg/mL (240 mg single dose) when Tecfidera was administered to healthy volunteers (FDA DMF Pharmacology Review). Dosing MS patients with 240 mg BID of Tecfidera resulted in a mean C.sub.max of 1.87 μg/mL and the AUC was 8.21 mg.Math.hr/L (Tecfidera label). The Biogen pharmacokinetics of Tecfidera showed high inter-subject variability with respect to the C.sub.max value.

(35) To achieve the required MMF concentration in the blood, 150 μg of MMF equivalent conjugate (PEG2000) was injected IV and SC routes and blood samples were collected up to day-10 and MMF concentrations were analyzed. The MMF levels were highest at day-1 in both cases and gradually decreased till day-7. However, no MMF was observed at 240 h (day 10) in either route of administration. At day-7 the MMF concentrations were 2.00 μg/mL (IV) and 2.36 μg/mL (SC). In the above findings the MMF concentration observed were comparative to those observed with therapeutic dose of oral Tecfidera 240 mg twice daily.

(36) A full-scale pharmacokinetics study was done using MMF conjugate (PEG2000) with SC route of administration. Two doses strength of the MMF conjugate were injected (100 and 150 μg of MMF equivalent) subcutaneous in 5 weeks BALB/C mice (n=5) and the MMF concentrations were determined up to Day 10. The objective of the study was to determine the pharmacokinetics of active drug MMF following subcutaneous injection, with the goal of achieving plasma concentrations in the 1-10 μg/mL range. The MMF concentration was determined with HPLC and PK parameters such as t.sub.1/2, C.sub.max, T.sub.max, AUC, CL (clearance), and Vd (volume of distribution) were calculated.

(37) The pharmacokinetics results are shown in FIG. 6. It was observed that the 100 μg of MMF equivalent conjugate dose resulted in MMF plasma concentrations ranging from 7.7 μg/mL (6 h) to 1 μg/mL (day 7) with Cmax concentration at 12 h (9.4 μg/mL). In case of 150 μg equivalent of MMF, the plasma concentrations were ranging from 15.5 to 1.67 μg/mL when monitored starting from 6 h to day-7. The MMF was not detected at day-10 in either dose groups. The results suggest that there is a good correlation between administered dose and plasma concentrations, at least in this dose range. Other pharmacokinetic non-dose dependent parameters such as the volume of distribution, clearance and half-life showed good concordance for the two doses.

(38) Compositions of MMF-Polymer Conjugates

(39) In an embodiment, this invention is directed to an injectable dosage form, adapted for administration to a patient parenterally including subcutaneous, intramuscular, intravenous or intradermal injection. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

(40) Materials and Methods

(41) Materials: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 4-(Dimethylamino) pyridine (DMAP) was purchased from Sigma-Aldrich (St. Louis, Mo., USA). mPEG-OH (1 kDa and 2 kDa) were procured from Creative PEGworks (Chapel Hill, N.C., USA). ACS grade dimethylformamide (DMF), dichloromethane (DCM), chloroform and methanol were obtained from Fisher Scientific. Regenerated cellulose (RC) dialysis membrane with molecular weight cut-off 1000 Da was obtained from Spectrum Laboratories, Inc. (Rancho Dominguez, Calif., USA). Deuterated chloroform (CDCl.sub.3) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, Mass., USA).

(42) Preparation of MMF-PEG1000 conjugate: Monomethyl fumarate (0.7 gm, 5.52 mmol) was dissolved in 30 mL of DCM/dry dimethylformamide (9:1, v/v) in a 100 mL two neck flask under nitrogen environment and EDC (1.4 gm, 9.2 mmol) and DMAP (0.03 g, 0.27 mmol) were added to it and stirred the reaction mixture for half an hour in an ice bath. Finally the mPEG-OH (1 kDa, 1.0 g, 0.92 mmol) was added dissolved in 10 mL of dry DCM and the reaction mixture was stirred for 24 h at room temperature. The solvent was evaporated at room temperature under vacuum. The crude product was purified using column chromatography over silica gel (60-120 mesh). The mixture of solvents was used (0.75% methanol in dichloromethane) as eluent afforded 0.52 g of MMF-PEG1000 conjugate (47% yield and Rf=0.6, 5% methanol in DCM). The final MMF-PEG1000 conjugate was characterized by reverse-phase HPLC, proton NMR and MALDI-TOF mass spectroscopy. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.33 (s, 3H, OCH.sub.3 of mPEG), 3.44-3.75 (m, backbone OCH.sub.2CH.sub.2O of mPEG), 3.79 (s, 3H, OCH.sub.3 of MMF), 4.35 (m, 2H, OCH.sub.2 of mPEG adjacent to MMF), 6.87 (m, 2H, —CH═CH— of MMF).

(43) Preparation of MMF-PEG2000 conjugate: Monomethyl fumarate (0.35 g, 2.74 mmol) was dissolved in 30 mL of dry DCM/dry DMF (9:1, v/v) under nitrogen condition in an ice bath; and EDC (0.69 g, 4.5 mmol) and DMAP (0.01 g, 0.13 mmol) were added to it. The reaction mixture was stirred for 30 min and mPEG-OH (2 kDa, 1 g, 0.45 mmol) dissolved in 10 mL of dry DCM was added to the reaction mixture. The resulting reaction mixture was stirred for 24 h at room temperature. The solvent was removed under reduced pressure at room temperature and the obtained crude product was dialyzed in DI water using dialysis membrane (MWCO 1 kDa) for 36 h. The obtained solution was lyophilized to get 0.72 g of MMF-PEG2000 conjugate (68% yield). The final MMF-PEG2000 conjugate was characterized by HPLC, .sup.1H NMR and MALDI-TOF mass spectroscopy. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.87 (s, 2H, —CH═CH— of MMF), 4.35 (m, 2H, OCH.sub.2 of mPEG adjacent to MMF), 3.81 (s, 3H, OCH.sub.3 of MMF) 3.43-3.76 (m, backbone OCH.sub.2CH.sub.2O of mPEG), 3.36 (s, 3H, OCH.sub.3 of PEG).

(44) Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR spectra of final conjugates as well as starting materials was recorded on a Varian Spectrometer (400 MHz). Tetramethylsilane (TMS) was used as internal standard and deuterated chloroform (CDCl.sub.3) was used as solvents to dissolve the conjugates.

(45) Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry: MALDI-TOF mass spectra was recorded in a AB-Sciex 5800 MALDI/TOF-MS instrument operating in the reflector mode. 2,5 Dihydroxybenzoic acid (DHB) was used as matrix and cytochrome c (MW 12361 g/mol) was used as external standard. The matrix solution was prepared by dissolving 20 mg of matrix in 1 mL of deionized water/ACN (0.1% TFA; 1:1). Samples were prepared by mixing 10 μL of conjugates (2 mg/mL in methanol) with 100 μL of matrix solution, and subjected 1 μL of sample mixture onto the MALDI plate. The samples were allowed to air-dry at room temperature and used for analysis.

(46) High performance liquid Chromatography (HPLC): The MMF-PEG conjugates were analyzed by system gold HPLC instrument (Beckman Coulter. Inc. Brea, Calif.) equipped with binary pump, UV detector, and autosampler interfaced with 32 Karat software. Acetonitrile:phosphate buffer pH 6.8 (75:25, v/v) in gradient flow (1 mL/min) was used as the mobile phase and chromatograms were monitored at 210 nm. Supelcosil LC-18 column with 5 μm particle size, 25 cm length, 4.6 mm internal diameter was used for the analysis.

(47) Drug Release Study in PBS: The drug release study of the MMF-PEG conjugates was performed in 0.1 M phosphate buffer (pH 7.4) at 37° C. A concentration of 3 mg/mL of the conjugate was placed in a water bath and the temperature of the bath was maintained at 37° C. with constant mixing. Samples were collected at appropriate time points and lyophilized sing liquid nitrogen. The MMF-PEG conjugates along with released MMF were extracted from the lyophilized powder using acetonitrile and the samples were analyzed in reverse-phase HPLC. The peaks of MMF-PEG conjugates and MMF were monitored at 210 and 260 nm respectively. Since the MMF also further degrades, peak area of MMF-PEG conjugate was used for the analysis.

(48) Drug Release Study in Plasma: The MMF-PEG conjugates (3 mg/mL) were incubated in human pooled plasma diluted to 80% with 0.1 M PBS with constant mixing in water bath at 37° C. The plasma samples were collected at periodic intervals and lyophilized using liquid nitrogen. Both MMF-PEG conjugates and released MMF were extracted from the lyophilized samples using methanol. The samples were analyzed by reverse-phase HPLC using acetonitrile:phosphate buffer pH 6.8 (75:25) as mobile phase at 210 and 260 nm. The peak area of MMF was used for the analysis.

(49) To obtain a standard plot, MMF was accurately weighed and spiked in blank plasma. Stock solution of 5000 μg/mL was prepared and was further diluted to 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.9 and 0.9 μg/mL (n=2). A standard plot was obtained with regression equation y=260748x+371377 (Correlation coefficient=0.9976). Proteins were precipitated using 2M trichloroacetic acid and centrifuged at 3000 rpm at 4° C. for 10 min. The supernatant was transferred into a fresh 1.5 ml tubes and freeze dried. MMF was extracted from freeze dried samples using acetonitrile and centrifuged at 3000 rpm at room temperature for 10 min. Supernatant was analyzed by HPLC using acetonitrile:phosphate buffer pH 6.8; 75:25, as a mobile phase. The calibration plots were constructed between the absorbance values and the respective concentration values. MMF peak was monitored at 210 nm.

(50) Dose and route determination studies of the MMF conjugates in mice. A pharmacokinetic pilot study was performed with five to six-week-old BALB-NeuT mice (n=2). Each animal was injected intravenously or subcutaneously with a single dose of MMF-PEG conjugates (500 μg and 150 μg of MMF equivalent conjugates) in a total 200 μL of PBS. Mice were euthanized at different time intervals post-injection and blood samples were withdrawn via cardiac puncture. Plasma were collected from the samples by centrifugation at 3000 rpm at 4° C. for 20 min. The proteins were precipitated using 2M trichloroacetic acid and centrifuged at 3000 rpm at 4° C. for 10 min. The supernatant was transferred into fresh 1.5 ml tubes and freeze dried. The drug was extracted using acetonitrile and centrifuged at 3000 rpm at room temperature for 10 min. Supernatant was analyzed by HPLC using acetonitrile:phosphate buffer pH 6.8; 75:25, as a mobile phase. Peak area of MMF was used for the analysis.

(51) A full-scale pharmacokinetic study was performed with 5-week old BALB/c mice (n=5). Each animal was injected subcutaneously with a single dose of MMF-PEG2000 conjugate (100 μg and 150 μg of MMF equivalent conjugate) in a total 200 μL of PBS. Mice were euthanized at 6, 12, 24, 72, 120, 168, and 240 h post-injection and blood samples were withdrawn via cardiac puncture. Plasma were collected from the samples by centrifugation at 3000 rpm at 4° C. for 20 min. The proteins were precipitated using 2M trichloroacetic acid and centrifuged at 3000 rpm at 4° C. for 10 min. The supernatant was transferred into fresh 1.5 ml tubes and freeze dried. The drug was extracted using acetonitrile and centrifuged at 3000 rpm at room temperature for 10 min. Supernatant was analyzed by HPLC using acetonitrile:phosphate buffer pH 6.8; 75:25, as a mobile phase. Peak area of MMF was used for the analysis.

(52) TABLE-US-00001 ABBREVIATIONS Dimethyl fumarate (also used for N,N-dimethyl formamide) DMF Food and Drug Administration FDA Matrix Assisted Laser Desorption/Ionization MALDI Monomethyl fumarate MMF Multiple sclerosis MS Nuclear Magnetic Resonance NMR Phosphate Buffered Saline PBS Polyethylene glycol PEG relapsing-remitting multiple sclerosis RRMS N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide EDC•HCl hydrochloride 4-(Dimethylamino)pyridine DMAP Dichloromethane DCM

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