2 4 6 8 10 12 14

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Abs

orba

nce

at 2

35nm

(AU

)

pH

Physical and Chemical Properties of Boronic Acids: Formulation ImplicationsWilliam A. Marinaro and Valentino J. Stella

Department of Pharmaceutical Chemistry, The University of Kansas

BackgroundBackgroundEffect of Polyols on Boronic Acid pKa Values Effect of Polyols on Boronic Acid pKa Values

ConclusionsConclusions

PurposePurpose SolubilitySolubility

ReferencesReferencesAcknowledgementsAcknowledgements

Boronic acid compounds are the focus of increasing interest as therapeutic agents due to their ability to inhibit enzyme activity, among other characteristics. Many peptide boronic acid compounds have been investigated as enzyme inhibitors with implications in pathologies as varied as Alzheimer’s disease, cervical cancer, blood clotting disorders, hepatitis C, etc. (1). In 2003 the drug Velcade® (bortezomib), figure 1, was approved by the FDA as a second-line treatment for multiple myeloma, it acts via inhibition of the 26S proteasome in mammalian cells (2). As shown in scheme 1, the role of the boronic acid moiety in this inhibition is to interact with the hydroxyl of the N-terminal threonine of the β-subunit of the proteasome (3).

1. W. Yang, X. Gao, and B. Wang. Boronic acid compounds as potential pharmaceutical agents. Med Res Rev 23: 346-68 (2003).

2. Millennium Pharmaceuticals. VELCADE® (bortezomib) for Injection PRESCRIBING INFORMATION, Cambridge, MA, 2004.

3. T. Mc Cormack, W. Baumeister, L. Grenier, C. Moomaw, L. Plamondon, B. Pramanik, C. Slaughter, F. Saucy, R. Stein, F. Zuhl, and L. Dick. Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. J Biol Chem 272: 26103-9 (1997).

4. K. Yoshino, A. Suzuki, Y. Mori, H. Kakihana, C. Honda, Y. Mishima, T. Kobayashi, and K. Kanda. Improvement of solubility of p-boronophenylalanine by complex formation with monosaccharides. Strahlentherapie und Onkologie: Organ der Deutschen Rontgengesellschaft. [et al] 165: 127-9 (1989).

5. L. Plamondon, L. Grenier, J. Adams, and S.L. Gupta. Formulation of Boronic Acid Compounds. United States Patent. Patent No. US 6,699,835 B2, March 2, 2004.

6. A. Albert & E.P. Serjeant. The Determination of Ionization Constants: A Laboratory Manual. London, UK: Chapman and Hall Ltd.; 1971.

While the pharmacologic properties of these compounds appears to be impressive, there are obstacles to their formulation into suitable dosage forms. One obstacle is their apparent low solubility. Workers have

observed solubility values for boronic acid containing compounds well below that of the respective non-boronic acid containing analog (4). It was also noted that solubility could be increased by adding monosaccharides to the aqueous solutions (4). Further, the formulators of Velcade® observed an increase in the solubility of the drug when it was lyophilized in the presence of mannitol (5). They attributed this to the formation of boronic acid esters, shown in green, and the simultaneous avoidance of forming the less soluble trimeric boroxine species shown in orange (Figure 2). A further explanation of this increased solubility is a pKa lowering effect that polyols impart to boronic acids (Figure 3). The mechanism by which polyols help solubilize boronic acid containing compounds is not completely understood and deserves further study.

The authors would like to acknowledge; Alana Toro-Ramos for her assistance in developing the methodology for pKa determination by UV spectrophotometry, KaisaKinnari for her assistance in the UV spectrophotometry studies, and Dr. Richard Prankerd for his guidance and training in the ITC experiments.

This research was financially supported by:

•The Takeru Higuchi Predoctoral Fellowship•The NCI Predoctoral Training Grant•The Takeru Higuchi–Nigel Manning Intersearch Program •The Department of Pharmaceutical Chemistry, University of Kansas

A large change in the UV spectra of 4-MBBA is observed at 235nm, this was utilized in pKa determination by UV spectrophotometric titrations (Figure 7).

Figure 10 demonstrates the correlation between polyol size and pKa lowering effect. As the number of hydroxyls on the polyol increases, the effect on 4-MBBA increases. The greatest effect comes from mannitol and xylitol, as the pKa lowering effect plateaus at around pKa 6.2, almost a full 3 units below the original pKa of ~9.2.

•The solid state of boronic acids and boronate esters will be studied, with a particular emphasis on how to exploit the formulation of boronic acid containing drugs with polyols in order to maximize the binding phenomena.

•The solubility and dissolution of boronates will be explored in greater detail to further elucidate the mechanism of boronate ester and boroxine dissolution to yield free boronic acids.

Scheme 1

UV Spectra of 4-MBBA, Neutral and Anion

00.20.40.60.8

11.21.41.6

190 210 230 250 270 290

Wavelength (nm)

Abs

orba

nce

(AU

)

NeutralAnion

Figure 7

pKa = 9.15

Figure 8

Apparent pKa of 4-MBBA Vs. Polyol Concentration

5.96.4

6.97.4

7.98.4

8.99.4

0 0.1 0.2 0.3 0.4 0.5 0.6

Polyol Concentration (M)

4-M

BB

A A

ppar

ent p

Ka

GlycerinErythritolXylitolMannitol

Glycerin Erythritol Xylitol MannitolHO

OHHO

OH

OHHO

OH

OH

OH

HO

HOOH

HO

HO OH

HO OH

OH

OB

OBO

BR

RRsolid

BO

O

OH

OHOH

OH

Rsolid

RB

OH

OH

RB

OH

OH

< 1mg/mL solution 6 mg/mL solution

Figure 2

BR

OH

OH

HO

R

HO+ BR

O

R

O

boronic acidpKa: 9-10

boronate esterpKa: ~6

Figure 3

NH

HN B

OHN

N

O

O

OHFigure 1

Structure of Velcade® (bortezomib)

Future WorkFuture Work

Solubility of 4-MBBA Vs. pH

0.1

1

10

1000.00 2.00 4.00 6.00 8.00 10.00 12.00

pH

Solu

bilit

y (m

g/m

L)

No Mannitol (solubility mg/mL) 500mM Mannitol (solubility mg/mL)

Solubility Vs. 1/[H+]

0

5

10

15

20

25

30

35

0.0E+00 5.0E+09 1.0E+10 1.5E+10

1/[H+] (M-1)

Solu

bilit

y (m

g/m

L)

No Mannitol (solubilitymg/mL)

][][][ 0

0 ++=HHAKHAS a

pKa’: 8.69

Solubility Vs. 1/[H+]

0

5

10

15

20

25

30

35

0.0E+00 2.0E+07 4.0E+07 6.0E+07 8.0E+07

1/[H+] (M-1)

Sol

ubili

ty (m

g/m

L)

500mM Mannitol(solubility mg/mL)

pKa’: 6.39

pKa’: 5.87

The titration of 4-MBBA is shown (Figure 8). The curve is fit to a modified Henderson-Hasselbach equation.

Potentiometric titrations were also performed using a Mettler-Toledo DL-53 autotitrator and the pKa values were calculated as described by Albert and Serjeant (6). Figure 9 shows the type of data generated, in this case 4-MBBA alone yields a pKa of 9.31±0.01.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

7 7.5 8 8.5 9 9.5 10 10.5 11

pH

Titr

ant V

ol./1

Equ

iv. V

ol.

Figure 9

Figure 4 demonstrates the large effect mannitol has on the solubility of 4-MBBA. Highlighted in green is the effect at physiologic pH, a 10-fold increase in solubility. Figures 5 and 6 illustrate the direct effect on the pKa as determined from the solubility pH profile, as described by equation 1. Through figure 6 one can see that the ratio of mannitol to boronic acid has an effect on solubility through pKa.

Isothermal Titration Isothermal Titration CalorimetryCalorimetry

Apparent pKa of 4-MBBA Vs. Polyol Concentration by Potentiometric Titration

5.50

6.00

6.50

7.00

7.50

8.00

8.50

9.00

9.50

0 0.1 0.2 0.3 0.4 0.5 0.6

Polyol Concentration (M)

App

aren

t pK

a

GlucoseMannitolFructose

Apparent pKa of IBA Vs. Polyol Concentration by Potentiometric Titration

7.508.008.509.009.50

10.0010.5011.0011.5012.00

0 0.1 0.2 0.3 0.4 0.5 0.6

Polyol Concentration (M)

App

aren

t pK

a GlucoseMannitolFructoseSorbitol

UV Spectrophotometric Titration

HO

HO OH

HO OH

OH

Sorbitol

O

HO OH

HO OH

HO

Glucose

OH

OH

OH

HH

OHH

OHH

HO

HO

OH

HO OH

HOO

Fructose

OH

CH2

H

CH2

OH H

H OHO

HOHO

Figure 10 Figure 11 Figure 12

Figure 11 illustrates the utility of the potentiometric technique, described above, in studying the effect of glucose and fructose on the apparent pKa of 4-MBBA. The UV spectrophotometric technique could not be used due to spectral interference, most likely the carbonyl group. The mannitol experiment confirms that both the UV spectrophotometric and potentiometric titration techniques yield the same results, when accounting for the different concentrations used in the experiments. Fructose decreased the apparent pKa of 4-MBBA slightly greater than did mannitol, and much greater than did glucose.

Figure 12 applies the potentiometric technique to the study of IBA an alkyl boronic acid. It is critical to demonstrate that this phenomenon occurs with alkyl boronic acids, because most boronic acid containing drugs are alkyl, not aryl boronic acids. First, this series of experiments shows that a similar drop in pKa occurs, roughly 3 units or greater for the highest binding polyols, when alkyl or aryl boronic acids form boronate esters. Second, the rank order of the polyols’ effect is maintained.

Figure 4 Figure 5 Figure 6

BHO

HOO

4-Methoxybenzeneboronic acid(4-MBBA)

BHO

OH

Isobutylboronic acid(IBA)

Structure 1 Structure 2

The objective of the following research is to determine the effect of various polyols on the physical and chemical properties of two model boronic acids; 4-methoxybenzeneboronic acid (4-MBBA) shown in structure 1, and isobutylboronic acid (IBA) illustrated in structure 2. The research will quantify the ability of

the polyols, first, to form boronate esters with the model compounds. Second, to determine the effect that this formation has on the pKa and solubility of the boronate compound. This information should provide crucial insights to the formulation chemist when developing boronic acid containing pharmaceuticals.

0 1 2 3 4 5 6-4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Data: ManVar013_NDHModel: OneSitesChi^2/DoF = 1527N 1.11 ±0.0328K 2.43E3 ±158ΔH -5269 ±202.9ΔS -2.18

Molar Ratio

kcal

/mol

e of

inje

ctan

t

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0-4

-2

0

-20

-15

-10

-5

0

-10 0 10 20 30 40 50 60 70 80 90100110120130140150160170180

Time (min)

µcal

/sec

Molar Ratio

kcal

/mol

e of

inje

ctan

t

Figure 13

Figure 14

Figure 15

[ ] [ ] [ ] [ ]( ) [ ]⎥⎥⎦

⎤

⎢⎢⎣

⎡

⋅⋅⋅+⋅−⋅⋅+−⋅⋅+

+⋅Δ⋅=K

LKKLKnMKnMLHVQ

2411 2

Where:Q is the total heatΔH is the enthalpy of bindingK is the binding constantn is the number of binding sitesV is the volume of the reaction cell[L] is the ligand concentration[M] is the receptor concentration

Equation 2

Equation 1

Figure 15

ITC Instrumental Diagram

Figure from User’s Manual Revision 12705, Calorimetry Sciences Corp., Lindon, UT

30mM Mannitol Titrated into 1mM 4-MBBA at pH 11.6

30mM Mannitol Titrated into 1mM 4-MBBA at pH 11.6

Isothermal titration calorimetry or ITC, is a calorimetric analytical techniques which measures the heat evolved or absorbed upon titrating a solution of ligand into a solution of receptor. The instrumental setup is shown in figure 13. As the mole ratio increases during the titration and there is less free receptor, the total heat per injection decreases, as shown in figure 14. This profile is described, for a molecule with a single set of receptors, by equation 2. Thus, the data may be fit, as in figure 15, and one may solve for the variables: N, K, ΔH, and ΔS

The ITC data confirms the rank order of polyol effects observed during pKa lowering studies. Further, the binding constants observed are on the same order as those predicted from the pKa lowering studies.

•pKa determination by UV spectroscopic and potentiometric titrations have identified that some high binding polyols decrease the pKa of a boronate by over three units. The high binding polyols include some common pharmaceutical excipients.

•This effect occurs in both aryl and alkyl boronic acids, and is maintained regardless of the starting pKa of the unbound boronic acid. Further, the rank order of polyol effect is maintained from alkyl to aryl boronic acids.

•Isothermal titration calorimetry confirms that a boronic acid-polyol binding correlates with this pKa lowering effect.

•Solubility studies show that pKa lowering of boronic acids by polyols has a large effect on the solubility of these molecules, especially near physiologic pH.

Peptide BOH

OH

PharmacophoreHO Peptide B

OHPharmacophore

O

NANA-24.3±NA-26.7±0.6-21.5±1.9ΔH (kJ/mol)

±SD

NANA0.84±NA1.07±0.081.05±0.00n ±SD

13.2±0.823.7±0.7118±NA6254±6052767±39K (M-1) ±SD

2,3-butanediolglycerolerythritolsorbitolmannitolLigand:

NANA-24.3±NA-26.7±0.6-21.5±1.9ΔH (kJ/mol)

±SD

NANA0.84±NA1.07±0.081.05±0.00n ±SD

13.2±0.823.7±0.7118±NA6254±6052767±39K (M-1) ±SD

2,3-butanediolglycerolerythritolsorbitolmannitolLigand:

Interaction of various polyols with 4-methoxybenzene boronic acid as the receptor in ITC experiments