Discovery of Potent, Selective and Short-Acting Peptidic V2 Receptor

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Discovery of Potent, Selective and Short-Acting Peptidic V Receptor Agonists Kazimierz Wisniewski, Steve Qi, John Kraus, Brian Ly, Karthik Srinivasan, Hiroe Tariga, Glenn Croston, Erin La, Halina Wisniewska, Carlos Ortiz, Regent Laporte, Pierre Riviere, Gebhard Neyer, Diane Hargrove, and Claudio D. Schteingart J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00132 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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Journal of Medicinal Chemistry

Discovery of Potent, Selective and Short-Acting Peptidic V2 Receptor Agonists Kazimierz Wiśniewski,* Steve Qi, John Kraus, Brian Ly, Karthik Srinivasan, Hiroe Tariga, Glenn Croston, Erin La, Halina Wiśniewska, Carlos Ortiz, Régent Laporte, Pierre J-M. Rivière, Gebhard Neyer, Diane M. Hargrove, and Claudio D. Schteingart Ferring Research Institute Inc., San Diego, CA 92121, USA

1 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT. The vasopressin analogue desmopressin (dDAVP, 1) is a potent V2 receptor agonist approved in many countries for the treatment of diabetes insipidus, primary nocturnal enuresis, nocturia and coagulation disorders. Since 1 is primarily excreted via the kidneys, an age-related decline in kidney function leads to slower elimination, prolonged antidiuresis and hyponatremia. In search of novel, potent, selective and short-acting peptidic V2R agonists, we synthesized a series of C-terminally truncated analogues of [Val4]dDAVP, 2, modified in positions 2, 3, 7 and/or at the disulfide bridge. The peptides were evaluated for in vitro potency at the hV2 receptor, selectivity versus the related receptors (hV1aR, hV1bR, hOTR) and pharmacokinetic profiles in rodents and other higher species. The truncated analogues show excellent potency at the V2R, increased CL and shorter half-life in rats. Two compounds 19 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-Agm) and 38 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-D-ArgNEt2) have been selected for clinical development for nocturia.


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INTRODUCTION. Arginine vasopressin, AVP, acts on the vasopressin V2 receptor subtype expressed on principal cells in the collecting ducts of the kidney to induce translocation of aquaporin 2 (AQP2) channels to their apical membrane. This drives an increase in the reabsorption of free water resulting in the production of more concentrated urine and water conservation. Secretion of AVP by the pituitary gland in response to an increase in plasma osmolality is the primary determinant of water homeostasis in animals and humans.1 Its synthetic analogue desmopressin, (desamino-D-Arginine8 vasopressin, dDAVP), 1,2 is the only antidiuretic peptide available on the market to treat conditions where the secretion of endogenous AVP hormone is deficient or absent. Compound 1 is a potent V2 agonist, more selective and devoid of V1aR mediated pressor effects, and with longer plasma half-life than AVP, which makes possible its therapeutic use by bolus administration by the intravenous, oral, and intranasal routes. Since its discovery and first approval in 1972 for diabetes insipidus, 1 has gained approval over time for the treatment of conditions affecting much larger numbers of patients such as primary nocturnal enuresis (PNE)3 and nocturia.4 In addition, 1 is approved for the treatment of coagulation disorders including haemophilia A and von Willebrand’s disease.5, 6 Compound 1 is generally well tolerated, with most reported adverse effects related to hyponatremia. Hyponatremia is defined as a serum sodium concentration of less than 135 mmol/L7, but clinically significant hyponatremia is observed with sodium serum levels below 130 mmol/L.8 Symptoms of mild hyponatremia are headache and nausea, but in severe cases they can progress to confusion, seizures, and coma.9 It is thought that V2 agonists cause dilutional hyponatremia, which requires two factors to be present simultaneously: protracted duration of antidiuretic activity, that does not allow sufficient time for the kidneys to excrete accumulated water and higher intake of fluids than necessary for the condition.10 PNE is a



Page 4 of 53

multifactorial condition, with a significant polyuria component in many patients. In normal children, plasma concentrations of AVP increase during the night, causing urine concentration and decrease in urine flow to below the functional capacity of the bladder. Polyuric PNE patients appear to lack this diurnal rhythm in the secretion of AVP; thus, nocturnal urine production exceeds functional bladder capacity, and because they do not wake up they experience enuresis.11 Treatment with an appropriate dose of desmopressin before bedtime restores urine concentration for the duration of the night, with no or little effect expected during the following morning due to washout of the drug.12 In children treated with desmopressin for PNE the incidence of hyponatremia is very low.10 The small number of serious cases appear to have occurred due to the presence of intercurrent illnesses, other medications, longer plasma half-life, or unexpectedly higher drug bioavailability, combined in many cases with documented excessive intake of fluids.13 Nocturia is defined as the complaint of waking up to void one or more times per night. Its prevalence increases with age, and when the frequency is two or more voids per night it becomes bothersome due to sleep fragmentation. This may result in reduced quality of life, mood disturbances, poorer overall health, and an increase in falls and fractures.14 The causes of nocturia are multifactorial but a strong association with nocturnal polyuria has been found15 and in many patients it can also be treated with desmopressin before bedtime, suggesting that they may also have insufficient release of AVP during the night.16 However, the incidence of clinical and subclinical hyponatremia in this older population is higher than in PNE, which has been mitigated by reduction of the dose and more careful evaluation of patients.8,

17, 18

Although

hyponatremia may also be multifactorial, one contributing factor may be extended duration of antidiuretic action in the elderly population (together with inappropriate fluid intake).

The


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elimination of 1 is primarily via passive glomerular filtration into urine by the kidneys,19 and renal impairment or age-related decline in kidney function (glomerular filtration rate, GFR) might lead to slower elimination and extended half-life,

19, 20

which may result in prolonged

antidiuresis, and ultimately hyponatremia.10, 20 The pharmacokinetic profile of 1 in humans has been extensively studied.19, 21-24 In healthy subjects the systemic CL of 1 was determined to be 7.1 L/h (1.7 mL/kg/min for 70 kg) with t1/2el of 2.8 h.25 In patients with severe renal impairment the CL can be reduced to as low as 0.69 mL/kg/min and its elimination half-life lengthened to as long as 10 h.19 With the intent of providing antidiuretics with an improved profile in the elderly population we launched a drug discovery program to identify potent and selective V2 agonists with shorter half-life than 1 and elimination mechanism substantially less dependent on passive glomerular filtration by the kidneys. Small molecule V2R agonists have been reported in the literature. Despite the promise of having V2R agonists with considerable oral bioavailability, the small molecules VNA-93226 and OPC-5180327 have not advanced beyond Phase 1 in clinical trials and their current status is unknown. Two other small molecule compounds appear to be advancing at a slow pace: fedovapagon (VA106483)28 is currently undergoing a phase 2b/3 study to treat nocturia in men with benign prostatic hyperplasia (BPH) (https://clinicaltrials.gov/ct2/show/NCT02637960) and ASP-703529

is

in

a

phase

2

clinical

trial

for

nocturnal

polyuria

(https://www.clinicaltrialsregister.eu/ctr-search/search?query=2013-003701-25). Given the modest clinical progress with small molecule V2R agonists and their potential for drug-drug interactions in a population simultaneously treated with a considerable number of other drugs, we decided to undertake a peptide based drug discovery program.



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In reporter gene assays at human receptors, 1 is a very potent V2 agonist, fairly selective against the oxytocin and V1b receptors, and completely devoid of V1a activity. Its rat plasma protein binding is low and its CL in rats is 7.5 mL/kg/min, which is similar to the GFR in rats (~ 9 mL/kg/min)30, suggesting that 1 is cleared mostly by passive glomerular filtration in these animals, confirmed by the very low proportion of non-renal clearance remaining in nephrectomized rats (Table 1). a

a

HO

HO

O O 2

NH

N H

HN 1

4

O

O NH2

H N

2

NH

N H

HN

N

HN 5

O

O

O

O

N H

O

H N

7 8

O

9

O NH2

S

1

6

NH 4

O

NH2

H N

NH2 NH

S

3

6

NH

O

NH

S

3

O

S

O

NH2

N

HN 5

O O

O

O

N H

O

H N

7 8

O

9

NH2

NH2

Figure 1. Structures of reference compounds 1 (left) and 2 (right). Sequence positions are numbered at α-carbons In–house experience revealed that conservative amino acid modifications of 1 resulted in peptides with rat CL similar or lower than rat GFR (data not shown). We concluded that to obtain peptides with higher CL in rats and humans we would need to introduce more severe modifications in 1 in the hope of adding extra-renal mechanisms of clearance, while preserving potency and selectivity at the hV2 receptor. SAR studies to identify peptidic analogues of 1 with improved pharmacological profiles have been reviewed in the literature.31, 32 The Val4 analogue, [Val4]dDAVP, 2, has been reported to be more potent and selective than 1 in vivo rat models.33 Modifications of position 2 with bulky Land D-β,β-diphenylalanine resulted in potent and selective V2R agonists in rat in vivo.34, 35 The replacement of Phe3 with Thi has been reported to increase potency at the V2R as compared to 6 ACS Paragon Plus Environment

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2.36 Manning et al. investigated the impact of the C-terminal Gly residue removal on the antidiuretic activity of 1, 2, and related peptides in rats. The corresponding desglycine analogues, especially the ones with the C-terminal amide function preserved, retained only 10 - 50% of the antidiuretic activity of their parent compounds.37-40 Therefore, we designed and synthesized a series of C-terminally truncated analogues of 2 modified in positions 2, 3, 7 and/or at the disulfide bridge. The peptides were evaluated for in vitro potency in RGA assays at the human V2 receptor (hV2R), selectivity versus related receptors (hV1aR, hV1bR, hOTR) and systemic clearance in rat. The non-renal component of CL was assessed in bilaterally nephrectomized rats. Plasma protein binding (PPB) in rat plasma was determined by an ultracetrifugation method to help interpret the PK data. Allometric scaling of CL in several species was performed to attempt to predict CL in humans.41, 42 Selected analogues were tested for in vivo potency and duration of action in a rat antidiuresis model. We report here on the identification of two promising peptidic V2 agonists with different rat clearance and elimination half-lives as clinical candidates for the treatment of nocturia and other indications where short duration of antidiuretic action may be desirable.

RESULTS AND DISCUSSION. The reference peptides 1 and 2 were evaluated for their in vitro potency and selectivity in RGA assays at the human receptors and for PK parameters in rats (Table 1). The in vitro pharmacological profile of 1 determined in these assays was consistent with the literature data.43 Compound 1 was particularly selective vs. the V1aR (>1000-fold) presumably due to the desamino modification.44 Compound 2 was more potent as hV2R agonist and slightly more selective versus hV1bR than 1. 7 ACS Paragon Plus Environment


Table 1. Pharmacological profiles of reference peptides 1 and 2. hV2R EC50 (nM) (95%CI, nM)

hOTR EC50 (nM) (95%CI, nM)

hOTR Efficacy (%) (95%CI, %)

hV1bR EC50 (nM) (95%CI, nM)

hV1bR Efficacy (%) (95%CI, %)

Selectivity vs. hOTRa

Selectivity vs. hV1bRa

Rat iv CL±STD (mL/min/kg)b

Nonrenal CL (%)c

Rat PPB ±STD (%)

1

0.20 (0.19-0.21)

110 (60-200)

79 (61-97)

11 (6.5-20)

94 (86-101)

550

55

7.5±0.33

11

41±8.3

2

0.05 (0.01-0.20)

350 (220-570)

27 (20-34)

24 (1.1-554)

98 (54-140)

7000

480

9.6±0.87

NTd

31±6.3

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 53

No activity at the hV1aR up to 1000 nM; Compounds fully efficacious at the hV2R; a EC50 (receptor)/EC50 (hV2R). b 4 animals per compound were used. c Non-renal CL fraction is calculated as described in experimental section. The CL values in nephrectomized and shamoperated rats are given in Supplemental Information, Table S3. d NT – not tested

Our drug discovery program aimed at identifying new analogues with in vitro potency at the hV2R similar to 1 and 2 and improved selectivity vs. the related receptors (hV1a, hV1b and hOT), increased CL (>10 mL/min/kg) in rat, considerable non-renal clearance component (>50%), and predicted CL in humans from allometric scaling in the range of 3 – 6 mL/min/kg, which is approximately 1.5 - 3 times the CL of 1 in healthy subjects. Assuming that the physiological volume of distribution of the new peptides is similar to that of 1, i.e. extracellular fluid, this increase in CL would translate into decreased elimination half-life by a small factor to be determined experimentally. Since nonapeptides based on modified 1 tended to have similar CL to 1, we decided to explore C-terminal desglycineamides as lead series to find out whether they could provide compounds with higher CL and shorter half-life while maintaining potency at the hV2R. The removal of the C-terminal Gly residue had been reported to be tolerated by the rV2R as the corresponding analogues of 1 and 2 retained some activity (48% and 32%, respectively) in a rat antidiuretic assay.39, 40 Thus, the C-terminal glycine amide in 1 was replaced with various functionalities (Fig. 2, R3 = H, CH2OH or C(=O)-NHR4). To compensate for the expected loss of 8 ACS Paragon Plus Environment

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potency due to the truncation, the Phe3 residue was replaced with Thi

36

or Fpa and the Gln4

residue was fixed with Val as 2 was 4-fold more potent as a hV2R agonist than 1 in our preliminary studies in vitro (Table 1). In addition, the replacement of Gln4 with Val results in more lipophilic peptide 2 (Table S4, k’ 4.56 for 2 vs. 3.72 for 1) and slightly higher rat CL (9.6 mL/min/kg). In previous work with a family of similarly sized oxytocin analogues we had found that increased lipophilicity correlated with higher systemic clearance, even when it could be expected to also increase PPB.45 By fixing position 4 with Val we hoped that the increased lipophilicity of the analogues would result in higher CL values also in this series. For position 2 we employed conservative and more lipophilic replacements of Tyr (Cpa, Phe(4-Me) and Phe(4Et)). Since the Cpa analogue of desamino-arginine8 vasopressin (dAVP) was shown to be a weak antagonist of OTR in a rat uterotonic assay in vitro and was less active than the parent compound as V1aR agonist in a rat in vivo pressor assay,46 these Tyr replacements appeared to be a good strategy to improve selectivity vs. the related receptors. The modifications explored in this study, compounds 3 – 43, are summarized in Fig. 2. a

R1

O O 2

NH

Ar

N H

Y

3

O

6

NH 4

O

H N

R2

X

1

NH

Z

N

HN

7

5

O

O

O

N H

* 8

R3

O NH2

Figure 2. Modifications of 2 explored in this study. Sequence positions are numbered at αcarbons



Page 10 of 53

Analogues 3 – 43 were synthesized by a combination of solid and solution phase chemistry. The linear precursors of 3 – 10, 19, 38, 39, 41 – 43 were assembled on H-Aaa-O-2-ClTrt resins by standard Fmoc chemistry using DIC/HOBt mediated couplings. The carba thioether modifications of the disulfide bridge (X or Y = CH2, Fig. 2) were introduced to the peptide sequence by coupling Fmoc-Cys((CH2)3-COOtBu)-OH or Fmoc-Hcy((CH2)2-COOtBu)-OH.47 Fully protected peptide C-terminal acids were cleaved from the resin with 30% HIPF/DCM. For compounds 3 – 10 the carboxylic group was reduced to the hydroxymethyl group using the mixed anhydride method.48 For Agm compounds 38, 39, 41 – 43, the C-terminal acids were coupled with agmatine and for analogue 19 the linear fragment was coupled with H-D-Arg-NEt2. -The linear precursor of peptide 40 was assembled on 1,4-diaminobutane-2-ClTrt resin. After the cleavage from the resin, the C-terminal amino function was temporarily protected with the TFA resistant Z(2-Cl) group and the peptide was deprotected with TFA. The linear precursors of compounds 11 – 18, 20 – 37 were synthesized on BAL resin which was reductively aminated with an appropriate primary amine prior to the peptide assembly (Fig. 3). The peptides were cleaved with concomitant side chain protecting group removal using the TFA/TIPS/H2O 95/2.5/2.5 cocktail. All peptides with the thioether modifications of the SS bridge were cyclized in solution using pseudo-dilution method49 and HBTU/NMM as coupling reagents. The partially protected, cyclic precursor of analogue 40 was treated with TMSBr/thioanisole/TFA (1/1/6)50 to remove the Z(2-Cl) group. The disulfide bridge containing peptides 11 and 14 were cyclized with iodine in 5% aqueous TFA. All peptides were purified by preparative HPLC and lyophilized.


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CO2-tBu i

O

ii

R4

N H

O HN Fmoc

Boc-Aaa2-Aaa3-Val-Asn

NH Pbf N

NH2 a R1

CO2H

2

3

H-Aaa -Aaa -Val-Asn

NH2

O v

NH

Z

O

O

H N

N H

NH

Ar

N

N H

NH

Z N

N H

O

O

NH2

R4 N

N H

O

O HN

X Y

iv

X Y

iii

N R4

Pbf N

O R

NH

4

O

O

N H

H N

X Y

R2 NH

Z N

HN O O

O

N H

H N

R4

O

NH2 1

(i) FMPB resin/R NH2/NaBH(OAc)3/DCE/TMOF; (ii) Fmoc-D-Arg(Pbf)OH/DIC/DCM; (iii) SPPS (DIC/HOBt, PIP);

(iv) TFA/TIS/H2O; (v) HBTU/DIPEA/DMF

Figure 3. Synthesis of peptides amides 11 – 18 and 20 – 37. Synthesis of other compounds discussed in the text (3 – 10, 19 and 38 – 43) is depicted in Fig. S2 and S3.

Table 2. Pharmacological properties of peptide alcohols 3 – 10 a

R1

O HN

O 2

NH

Ar

N H

1

O

Cys

NH Val

O

H N

NH

Y

3

NH2

X

N

HN

7

Asn

O

O

O

N H

*

OH

8

O NH2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Compound


Page 12 of 53

Pharmacological dataa

Structure R1

Ar

X

Y

C* config

hV2R EC50 (nM) (95%CI, nM)

hOTR EC50 (nM) (95%CI, nM)


Selectivityb vs. hV1bR

Rat iv CL±STD (mL/min/kg)c

Nonrenal CL (%)d

Rat PPB± STD (%)

3

OH

2Thi

CH2

S

S

0.10 (0.040.22)

230e (100540)

520 (1801500)

5200

8.1±0.77

NTf

15±3.2

4

OH

2Thi

S

CH2

R

0.08 (0.030.19)

160e (46-520)

58 (26-130)

720

6.9±0.83

NTf

26±1.5

5

Cl

2Thi

CH2

S

R

0.10 (0.040.27)

>10000g N/A

140 (51-390)

1400

4.0±1.7

NTf

80±1.8

6

Cl

2Thi

S

CH2

R

0.08 (0.030.21)

>10000g N/A

64 (11-360)

800

2.8±0.46

66

89±5.9

7

Cl

2Thi

CH2

S

S

0.14 (0.100.19)

>10000g N/A

380 (1401000)

2700

8.1±0.43

NTf

81±0.53

8

Cl

Fpa

CH2

S

R

0.35 (0.121.1)

>10000g N/A

220 (95-530)

620

3.8±0.14

NTf

93±0.23

9

Me

2Thi

CH2

S

R

0.39 (0.230.66)

>10000g N/A

810 (2502600)

2000

5.3±0.41

30

68±0.69

10

Et

2Thi

CH2

S

S

0.35 (0.190.65)

>10000g N/A

>10000f N/A

>28000

9.4±1.3

80

84±0.29

a

No activity at the hV1aR up to 1000 nM. b EC50 (hV1bR)/EC50 (hV2R); c 4 animals per compound were used. d See footnote (c), Table 1. e Partial agonist. f NT – not tested. g No activity at the hOTR or hV1bR up to 10000 nM, the highest concentration tested. Efficacy values are given in Table S1.

The first chemical series we explored in this study were octapeptide alcohols (compounds 3 – 10, Table 2), in which the C-terminal group R3 (–C(=O)-Gly-NH2, Fig. 2) in compound 2 was replaced with the hydroxymethyl function and the analogues were also modified in pos. 2, 3 and


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at the disulfide bridge. The disulfide bridge modifications (X, Y) as well as the configuration of the *C chiral carbon in position 8 had very little impact on V2R potency in vitro (compare 3 - 7) but the carba-6 compounds 4 and 6 (X = S, Y = CH2) were less selective vs. hV1bR than their carba-1 counterparts 3, 5, 7 - 10. Analogues in this series were potent V2R agonists with 8 (Ar = 4-fluorophenyl), 9 and 10 (R1 = alkyl) being about 5-fold less potent than 2. Analogues 3 - 10 displayed improved selectivity versus both the V1bR (all partial agonists) and OTR except for the native Tyr2 compounds 3 and 4 (R1 = OH) that were less selective vs. hOTR than 2, as suggested by literature data.46 Since the replacement of the C–terminal group R3 with the hydroxymethyl group resulted in potent and selective hV2R agonists, we evaluated the compounds for their PK properties in male rats. Similarly to 1, the Tyr2 compounds (3, 4, R1 = OH) showed low PPB in rats whereas the 4-chloro analogues 6 – 8 exhibited surprisingly high binding. The 4-alkyl substituted analogues had intermediate PPB values. Despite their low free fraction (10000g N/A

>31000

11±0.78

81

88±1.1

12

2-thienyl

CH2

S

CH2

Et

0.29 (0.100.82)

170 (71-420)

5800

10±2.5

49

84±1.6

13

2-thienyl

S

CH2

CH2

Et

0.19 (0.090.38)

120 (49-310)

630

5.3±0.79

67

90±1.0

14

Ph

S

S

CH2

Et

0.38 (0.290.51)

>10000g N/A

>26000

7.1±0.42

47

95±0.30

15

Ph

CH2

S

CH(OH)

Et

0.29 (0.150.55)

150 (28-820)

510

3.3±0.19

NTh

91±0.53

16

2-thienyl

S

CH2

CH(OH)

Et

0.10 (0.040.28)

260 (70-960)

2600

4.0±0.53

58

90±0.86

17

2-thienyl

CH2

S

S

Et

0.10 (0.070.15)

160 (120220)

1600

28±12

80

93±2.3

18

2-thienyl

S

CH2

S

Et

0.11 (0.060.20)

40 (15-110)

360

11±1.2

NTh

92±0.84

19

2-thienyl

CH2

S

CH2

Etc

0.25 (0.140.44)

530 (420690)

2100

42±5.2

60

92±9.2

20

2-thienyl

CH2

S

CH2

Me

0.27 (0.140.44)

350 (170730)

1200

9.1±0.57

NTh

82±0.79

21

2-thienyl

CH2

S

CH2

Pr

0.33 (0.061.7)

250 (100620)

750

26±1.9

59

84±1.3

22

2-thienyl

CH2

S

S

Pr

0.21 (0.090.49)

120 (85-180)

570

31±11

90

83±0.85


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Compound


Structurea

Page 16 of 53

Pharmacological datab

Ar

X

Y

Z

R4 c



Selectivity vs. hV1bRd

Rat iv CL±STD (mL/min/kg)e

Nonrenal CL (%)f

Rat PPB± STD (%)

23

2-thienyl

CH2

S

CH2

i-Pr

0.45 (0.161.2)

580 (3301000)

1200

15±0.88

58

85±0.67

24

2-thienyl

CH2

S

CH2

cPr

0.23 (0.100.51)

480 (1901200)

2000

10±3.8

67

91±3.6

25

2-thienyl

CH2

S

CH2

CH2cPr

0.21 (0.130.34)

320 (160660)

1500

21±1.5

NTh

84±2.0

26

2-thienyl

CH2

S

CH2

Bu

0.26 (0.180.38)

190 (140250)

730

31±2.9

57

89±1.3

27

2-thienyl

CH2

S

CH2

i-Bu

0.22 (0.120.41)

210 (170270)

950

24±4.2

63

86±0.72

28

2-thienyl

S

CH2

CH2

i-Bu

0.22 (0.100.50)

>10000g N/A

>45000

NTh

NTh

NTh

29a

2-thienyl

CH2

S

CH2

i-Bu

0.29 (0.120.67)

>10000g N/A

>34000

51±1.9

NTh

84±2.4

30

2-thienyl

CH2

S

CH(OH)

i-Bu

0.23 (0.060.84)

480 (1301800)

2000

7.8±1.6

NTh

87±1.7

31

Ph

S

CH2

CH2

i-Bu

0.27 (0.100.68)

46 (10-210)

170

5.4±0.98

88

95±0.81

32

2-thienyl

S

CH2

CH(OH)

i-Bu

0.20 (0.090.46)

130 (50-360)

650

5.9±0.35

NTh

92±0.60

33

2-thienyl

CH2

S

CH2

Bn

0.19 (0.130.29)

150 (100220)

780

23±3.3

84

90±1.4

34a

2-thienyl

CH2

S

CH2

Bn

0.26 (0.210.32)

150 (82-270)

570

14±1.1

71

45±0.82


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound

Page 17 of 53

Structurea

Pharmacological datab

Ar

X

Y

Z

R4 c



Selectivity vs. hV1bRd

Rat iv CL±STD (mL/min/kg)e

Nonrenal CL (%)f

Rat PPB± STD (%)

35

2-thienyl

CH2

S

S

Bn

0.31 (0.200.49)

330 (160660)

1000

38±20

100

91±1.5

36

2-thienyl

CH2

S

S

EtThi

0.17 (0.120.24)

96 (73-130)

560

34±0.89

100

93±0.30

37

Ph(4-F)

CH2

S

S

EtThi

0.30 (0.160.56)

100 (96-110)

330

27±5.9

100

96±1.0

a

All compounds of R configuration at C* except for 29. Compounds 15, 16, 30, 32 contain Hyp (Z = CH(OH), the carbon atom of R configuration). R1 is Cl for all compounds except for 34 for which it is OH. b No activity at the hV1aR up to 1000 nM, no activity at the hOTR up to 10000 nM, the highest concentration tested for all compounds except for 35 (EC50 = 140 nM at 32% efficacy); Efficacy values at the hV1bR in the supporting info. c R5 = H for all compounds except for 19 where R5 = Et; d EC50 (hV1bR)/EC50 (hV2R). e 4 animals per compound were used. f See footnote (c), Table 1. g No activity up to 10000 nM, the highest concentration tested. h NT – not tested.

A wide range of total systemic CL values in the rat were observed in this alkyl amide series. Generally, rat CL increased with the increasing lipophilicity/number of carbon atoms in the alkyl groups. Within the compounds of the substructure c(Bua-Cpa-2Thi-Val-Asn-Cys)-Pro-D-ArgNR4R5 (12, 19 – 21, 23 – 27, 33) the methyl amide analogue 20 displayed the lowest CL (9.1 mL/min/kg) and the diethyl analogue 19 had the highest CL (42 mL/kg/min). Cyclic or branched alkyl groups resulted in lower CL than their straight alkyl equivalents (compare propyl peptides 21, 23 and 24 or butyl analogues 25 - 27). The modifications of the disulfide bridge had considerable effect on rat CL. As we observed in our previous study with OT analogues45 the carba-6 modification (X = CH2, Y = S) yielded compounds with lower CL than their carba-1 and



Page 18 of 53

disulfide bridged counterparts. For instance, for the carba-6 ethyl amide 13 the CL value was 5.3 mL/min/kg whereas its disulfide bridge analogue 11 and the carba-1 counterpart 12 showed higher CL values of 11 mL/min/kg and 10 mL/min/kg, respectively . In all cases where the pair was available, the carba-1 analogue had markedly higher CL than the corresponding carba-6 analogue (compare 17 and 18, 27 and 28 or 30 and 32). The Pro7 modification with a more polar amino acid Hyp (Z = CH(OH)) resulted in analogues with drastically reduced CL (compare 15 vs. 12 or 30 vs. 27) whereas the replacement with less polar Thz (Z = S) led in most cases to CL increases (e.g. 17 vs. 12 or 22 vs. 21) . Changing the configuration of the C-terminal Arg residue in compound 27 to S resulted in analogue 29 with the second highest CL in the series. Selected compounds were tested for non-renal contributions to CL in rats. All new analogues tested in the series showed high (>50%) extra-renal CL. Some compounds (35 – 37) were cleared exclusively by non-renal mechanisms (CLnr = 100%), a rather unusual phenomenon for small peptides. The presence of an aromatic ring in the amide alkyl as well as the sulfur atom in the pyrrolidine ring (Z = S) were the structural motifs facilitating high CLnr . All compounds in Table 3, except 34, had similar rat PPB, which was higher than 80%. Since the passive glomerular elimination rate should be proportional to the free fraction of the peptide it would be expected that its contribution to the total clearance in rats would be less than ~2 ml/kg/min (CLr = fu.GFR) We can rationalize the observed total rat CL values as arising from this residual kidney clearance of the free peptide plus the contribution of non-renal clearance, which clearly depends on the structure of the peptide, can be very high, and apparently not hindered by PPB. We note that removing the kidneys from the circulation in nephrectomized rats eliminates not only passive glomerular filtration but also the possibility of active kidney tubular excretion of any drug. However, to the best of our knowledge active tubular secretion of peptides has not 18 ACS Paragon Plus Environment

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been described in the literature. In this work we will refer to renal clearance as a synonym of passive glomerular filtration. The mechanisms of the extra-renal clearance of peptides 3 - 43 are not known at this time, but the most likely candidates are proteolysis and transport into organs. Other than the tendency for more lipophilic compounds to have higher total rat clearance and the carba-1 modification, a clear SAR correlation for CL is not immediately apparent. Lastly, we investigated if the substituent R3 in Fig. 2 is actually required to preserve high agonist potency at the hV2R (compounds 38 –43, Table 4) and the consequences of its removal on PK. To design such compounds we applied the SAR lessons learned with the hydroxymethyl and N-alkylamide series (3 – 37). In this subset, only the carba-1 modified analogues (X = CH2, Y = S in Fig. 2) were prepared. Somewhat surprisingly, the compounds in this series turned out to be potent hV2R agonists in vitro. Particularly, 38 was found to be very potent as a hV2R agonist (EC50 = 0.07 nM) and considerably more selective than 1 or 2 vs. the related receptors. Interestingly, we found that the guanidine function (R6 = C(=NH)-NH2) was not essential for hV2R activation. Analogue 40 in which the guanidine function was replaced with a less basic primary amine (R6 = H in Table 4) retained very good potency in vitro and displayed an excellent selectivity profile. All compounds where inactive at the hV1a and hOT receptors, including 41 which carries a Tyr2 (R1 = OH). CL values in this series were similar to the ones of small alkyl amide compounds (compare 38 with 12 or 20) and generally followed the trends observed in the previous series. As expected the Thz7 containing peptide 42 (Z = S) showed the highest CL (16 mL/min/kg) in the subset. Compound 40 displayed a similar PK profile (CL = 7.2 mL/min/kg, CLnr = 19%) to that of 1 which suggests that presence of the native Tyr2 (R1 = OH), which severely reduces PPB, should be avoided if a reduction in renal CL is desired.



Table 4. Structure and pharmacological properties of peptides 38 – 43 R

R1

O O 2

NH

Ar

N H

1

S

3

O

Cys

NH Val

H N

Z

N

HN

H N

7

Asn

O

O

O

O

N H

8

R6

O NH2

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 53

Pharmacological dataa

Structure R1

Ar

Z

R6



Selectivity vs. hV1bRb

Rat iv CL±STD (mL/min/kg)c

Nonrenal CL (%)d

Rat PPB±STD (%)

38

Cl

2Thi

CH2

C(=NH)NH2

0.07 (0.04-0.11)

110 (87-130)

1500

14±1.3

57

85±1.0

39

Cl

Fpa

CH2

C(=NH)NH2

0.31 (0.10-0.98)

140 (98- 210)

450

7.5±2.0

NTe

94±0.29

40

OH

2Thi

CH2

H

0.19 (0.08-0.46)

>10000f N/A

>52000

7.2±0.8

18

42±30

41

OH

Fpa

CH2

C(=NH)NH2

0.10 (0.03-0.35)

180 (120-260)

1800

8.7±0.52

45

31±20

42

Cl

2Thi

S

C(=NH)NH2

0.12 (0.08-0.20)

140 (94-210)

1100

16±4.2

90

84±0.79

43

Cl

Fpa

S

C(=NH)NH2

0.22 (0.10-0.45)

110 (96-140)

500

9.9±0.63

85

96±0.23

a

No activity at the hV1aR up to 1000 nM, no activity at the hOTR up to 10000 nM, the highest concentration tested for all compounds except for 40 (EC50 = 650 nM at 28% efficacy). b EC50 (hV1bR)/EC50 (hV2R). c 4 animals per compound were used. d See footnote (c), Table 1. e NT – not tested. f No activity up to 10000 nM, the highest concentration tested.

The SAR studies described above produced a relatively high number of compounds which are potent and selective at hV2R, have moderately high PPB, high total rat CL, and significant


Page 21 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


contribution from extra-renal CL, but with no easy way to identify those that might have human CL in the 3 – 6 mL/min/kg range we desired. We then decided to investigate whether allometric scaling41,

42

of CL might offer an avenue for selection. Compounds with no or low PPB and

exclusive elimination by passive glomerular filtration would be expected to exhibit reasonable allometric scaling of their CL because GFR itself scales allometrically.52 However, the compounds of our interest were designed and selected to have a low proportion of total CL from elimination by passive glomerular filtration, and a priori there was no expectation that the extrarenal component of elimination would also scale allometrically. Moreover, we had no evidence that the PPB of the peptides would be similar in various animal species and in humans. Rather than trying to dissect the various factors that could determine CL in animals and humans, we decided to attempt an empirical correlation of total CL vs body weight (BW) in rats (Tables 2 – 4), dogs, Cynomolgus monkeys, and pigs (Table S2 in Supplemental Info). The rationale being that if we found compounds with good allometric scaling of CL across four species they would also have a high probability of continuing the correlation to humans. This would increase our confidence that the value of CL extrapolated allometrically to humans might be observed in the clinic. We selected for this study compounds with rat total CL≥10 mL/min/kg, excellent potency/selectivity profile in vitro, and high (>50%) non-renal CL component in rats (11, 12, 17, 19, 21, 23, 24, 26, 27, 33 and 38), and two carba-6 peptides with low rat CL (16 and 31) as they might be informative on the scope of any emerging allometric scaling relationship. We excluded peptides with very high CL in rat (> 45 mL/min/kg, e.g. 29) as well as the few ones with very high fraction (≥85%) of non-renal CL in rat (e.g. 35 – 37, 42). Whole body CL was graphed as a function of body weight in log-log allometric scaling plots (Fig. 4) and the data fitted to the standard allometric equation:



Page 22 of 53

𝐶𝐿𝑤𝑏 = 𝑎 × 𝐵𝑊𝑏 The value of the allometric coefficient a and the exponent b were obtained from the corresponding trend lines from rat, Cyno, dog and pig CL data. The regression coefficient of the correlation R2 and the value of b were used as measures of goodness of the allometric correlation. The predicted values of whole body CL (CLwb) and the CL per kg body weight for humans were calculated assuming a BW = 70 kg (Table 5).

Figure 4. Allometric scaling for selected compounds. Panel A (compound 31, carba-6), B (11, disulfide), C (19, carba-1) and D (38, carba-1). The CL values used to construct the graphs are listed in Supplemental Info (Tables S1, S2). The CL values are represented by blue diamonds (rat), red dots (Cyno), orange squares (dog) and green triangles (pig). 3 animals per compound 22 ACS Paragon Plus Environment

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were used with the exception of rat experiments where 4 animals were used. Additional allometric scaling plots are shown in Fig. S1. Table 5. Allometric scaling parameters and predicted CL in humans for selected compounds Compound

aa

ba

R2

Whole body CL CL (predicted, (predicted, mL/min/kg)b mL/min)b

1

5.338

0.742

0.981

129

1.8

11

9.220

0.786

0.962

260

3.7

12

6.950

0.722

0.942

151

2.2

16

4.576

0.992

0.989

310

4.4

17

17.064

0.678

0.996

305

4.4

19

24.685

0.633

0.999

365

5.2

27

16.063

0.673

0.964

280

4.0

31

7.689

0.910

0.912

368

5.3

33

19.092

0.798

0.968

568

8.1

38

10.917

0.787

0.995

309

4.4

a

Allometry equation parameters. b For CL calculations 70 kg body weight was assumed. For corresponding graphs see Fig. 4 and S1

The values of R2 and visual inspection of the plots in Fig. 4 and Fig. S1 show that many compounds did not scale allometrically, e.g 11 and 31, while others did it very well, e.g. 19 and 38. Given the small number of peptides tested we could not establish an effect of the bridge substitution pattern (carba-1, carba-6, disulfide) on the correlation. However, the analogues with the best allometric scaling, 17, 19, and 38,

(Table 5, b0.98) have the carba-1

substitution. They also extrapolate to a predicted CL in humans in the 3 – 6 mL/min/kg range.



Page 24 of 53

With both 17 and 38 having the same predicted CL in humans (4.4 mL/min/kg), compound 38 was preferred over 17 due to its marginally better potency in vitro (Tables 2, 3). Therefore, two analogues: 19 (higher predicted CL in humans, Fig. 4, panel C) and 38 (lower predicted CL in humans, Fig. 4, panel D) were chosen for further evaluation in vivo. To confirm that the lead peptides were potent and short acting in vivo we tested the reference 1 and compounds 19 and 38 in a rat model of antidiuresis, which required knowing their potency at the rV2 receptor. The in vitro potency of the three compounds at the rat V2R was similar (Table 6), but substantially higher than at the human V2 receptor (Tables 1, 3, and 4). A constant intravenous infusion of the peptide was instituted in euvolemic rats to establish a known steady state plasma concentration (Css) and the antidiuretic activity was determined by measuring cumulative weight of urine output from 0 to 180 min. The infusion rate R0 required to establish each Css was calculated using the value of CL for the compound (Table 6) with the equation R0 = CL . Css. It was not possible to verify the established plasma concentrations Css experimentally as they are well below the limit of quantification of even modern LC-MS methods when only a small volume of blood sample is available. Table 6. Rat pharmacological profiles of compounds selected for in vivo testing Compound

rV2R EC50 (nM) (95%CI, nM)

rV2R Efficacy (%) (95%CI, %)

Rat iv CL±STD (mL/min/kg)a

Rat iv t1/2el±STD (min)a

Rat PPB±STD (%)

1

0.03 (0.03-0.04)

100 (N/A-N/A)b

7.5±0.33

40±5.2

41±8.3

19

0.05 (0.03-0.11)

91 (80-102)

42±5.2

6.4±0.65

92±9.2

38

0.02 (0.01-0.04)

99 (89-110)

14±1.3

16±3.0

85±1.0

a

4 animals per compound were used. b Reference compound. 24 ACS Paragon Plus Environment

Page 25 of 53

The results of the in vivo study are summarized in Fig. 5 and 6 and tabulated in Table 7. Analogues 19, and 38, infused at range of doses to achieve target Css of 0.3 – 100 pM, produced

6 Vehicle

Compound 1 Compound 19

4

2

0 0.1

1

10

Cumulative Urine Output at 180 Minutes (g)

dose-related decreases in urine output with similar maximal efficacy to 1.

Cumulative Urine Output at 180 minutes (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6

Compound 1 Compound 38

Vehicle

4

2

0

100

0.1

Target Css (pM)

1

10

100

Target Css (pM)

Figure 5. Comparison of in vivo antidiuretic potency and maximal effect of 1 and 19 (left graph) and 1 and 38 (right graph) in normal euvolemic rats assessed by measuring weight of cumulative urine output at 3h of intravenous infusion. Shown are mean ± SEM for urine output in N = 200 vehicle-treated animals and 15 - 20 compound-treated animals at each target Css (error bars that are not visible are encompassed within the symbol). Fitted parameters for dose-response are summarized in Table 7. Table 7. Dose-response analysis of compound 1, 19 or 38 effect on urine output at 3h of intravenous infusion in normal euvolemic rats Compound

ECp50 (Target Css, pM)

95% Top (g) Confidence Interval for ECp50 (Target Css, pM)

95% Confidence Interval for Top (g/3 h)

Maximal Effect (Bottom) (g/3 h)

95% Confidence Interval for Maximal Effect (g/3h)

1

0.32

0.28 – 0.40

5.2

5.0 – 5.3

0.99

0.91 – 1.1

19

0.43

0.10 – 1.8

5.0

3.7 – 6.4

1.0

0.26 – 1.8 25

ACS Paragon Plus Environment


Page 26 of 53

Compound

ECp50 (Target Css, pM)

95% Top (g) Confidence Interval for ECp50 (Target Css, pM)

95% Confidence Interval for Top (g/3 h)

Maximal Effect (Bottom) (g/3 h)

95% Confidence Interval for Maximal Effect (g/3h)

38

0.64

0.26 – 1.5

4.2 – 5.8

1.3

0.82 – 1.8

5.0

Reported are best-fit values and 95% confidence intervals from dose-response curve analysis. Potency was expressed as the ECp50, the effective plasma concentration of the compound causing half-maximal suppression of urine output; maximal effect to suppress urine output was reported as the Bottom in the dose-response fit. 16-20 animals were used at each target Css

When expressed in terms of effective plasma concentrations (ECp50) the potencies of 19 and 38 were also similar to 1. Notably, the values of in vivo ECp50 were in the subpicomolar range, whereas their in vitro potencies were in the 20-50 pM range (Table 6). This result was not unexpected as both AVP53 and 154 are known to have single digit pM potency in humans and animals. This appears to be a consequence of the exquisite downstream amplification of the effect relayed via the V2 receptor in the principal cells of the collecting ducts of the kidney, which is not fully reflected in artificial cell based assays. It is interesting to note that 1 is about a hundredfold more potent in vivo in rats than in the cell based assay, while 19 and 38 are only ten times more potent (Table 7 vs. Table 6). This may be due to the much higher PPB of 19 and 38 in rats than of 1. To investigate whether the higher CL of 19 and 38 in rats translated into a shorter duration of antidiuretic action compared to 1 we examined the time required to restore urine output after stopping an intravenous infusion of 19 and 38 at 180 min (Fig. 6). Vehicle-treated animals exhibited normal diuresis with increasing cumulative urine output throughout the 8 h time course. During the compound infusion period (0 - 180 min.), maximally effective doses of 1, 19,


Page 27 of 53

and 38 supressed urine output by ~80% compared to vehicle treatment. After stopping an infusion of 1 at a rate to target Css 30 pM the compound continued supressing urine output for an additional 5 h, as could be reasonably expected from its half-life of 60 min and its ECp50 0.32 pM in these animals. By contrast, after terminating an infusion of 19 to target Css 30 or 100 pM the diuresis recovered in about 1 h, in accordance with its much shorter 12 min half-life. After terminating an infusion of 38 targeted to Css 100 pM , diuresis recovered within about 1 h, but at a reduced urine production rate for the following hours, which can be rationalized by its intermediate half-life of 20 min and slightly lower potency ECp50 0.64 pM in rats. When starting from and end of infusion Css 30 pM the recovery of diuresis was almost immediate and at a faster urine production rate. We concluded that at least in this rat antidiuresis model compounds 19 and 38 are very potent antidiuretics with sub pM ECp50 values in spite of their higher PPB, and exhibit shorter duration of action than 1 in response to their higher CL and shorter half-lives.

vehicle

12

Compound 19 (30 pM)

10

Compound 1 (30 pM)

Cumulative Urine Weight (g)

vehicle

Cumulative Urine Weight (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound 19 (100 pM)

8 6 4 2

12

Compound 38 (30 pM) Compound 38 (100 pM)

10

Compound 1 (30 pM)

8 6 4 2 0

0 0

60

120

180

240

300

360

420

480

0

60

120

vehicle or peptide i.v. infusion Stop

180

240

300

360

420

480

Time (min)

Time (min)

vehicle or peptide i.v. infusion Stop

Figure 6. Rat antidiuretic time course at maximally effective target Css. Time courses of 19 (left graph) and 38 (right graph) (30 and 100 pM) compared to 1. Peptides were infused iv through 180 min. at doses to rapidly achieve and maintain the indicated target Css and cumulative urine weight was measured. At 180 min., peptide infusion was stopped and urine output was measured 27 ACS Paragon Plus Environment


Page 28 of 53

for an additional 300 min. Shown are mean urine output in N = 200 vehicle-treated animals and 16 - 20 compound-treated animals. Error bars (only shown at 60 minute intervals for clarity) indicate ± SEM in each treatment group.

CONCLUSION. A series of novel C-terminally truncated analogues of 1 and 2 with improved in vitro pharmacological profile and higher systemic clearance has been identified. These novel compounds retain the potent V2 receptor agonistic activity of 1 and display substantially improved selectivities vs. the related hV1aR, hV1bR, and hOTR as compared to 1. The C-terminal modifications described herein resulted in analogues with desirable PK profiles (CLanalogue >CL1, non-renal CL > 50%) and, in some cases, good allometric scaling in four species. However, as it would be unreasonable to expect that allometric scaling would predict the CL of these peptides in humans with any great accuracy, two compounds from different chemical series, 19 (FE 202217) and 38 (FE 201836), have been selected for clinical development as potential treatments for nocturia and other indications requiring short duration of antidiuretic action.

EXPERIMENTAL. Chemistry General. Amino acid derivatives were purchased from commercial providers (Aapptec, EMD Milipore, Bachem and Peptides International).

Resins were purchased from commercial

suppliers (PCAS BioMatrix Inc. and EMD Milipore). All additional reagents, chemicals and solvents were purchased from Sigma-Aldrich and VWR. Fmoc-Cys((CH2)3-COOtBu)-OH (the carba-1 derivative) and Fmoc-Hcy((CH2)2-COOtBu)-OH (the carba-6 derivative) were 28 ACS Paragon Plus Environment

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synthesized as described previously.47 Non-commercial H-Aaa-2ClTrt resins were prepared according to a literature procedure.55 Preparative HPLC was performed on a Waters Prep LC System using a PrepPack cartridge Delta-Pack C18, 300Å, 15 µm, 47 x 300 mm at a flow rate of 100 mL/min and/or on a Phenomenex Luna C18 column, 100Å, 5 µm, 30 x 100 mm at a flow rate of 40 mL/min. Analytical reverse phase HPLC was performed on an Agilent Technologies 1200rr Series liquid chromatograph using an Agilent Zorbax C18 column, 1.8 µm, 4.6 x 110 mm at a flow rate of 1.5 mL/min. Final purity of analogues was assessed on a 1100 Agilent Liquid Chromatograph using the following analytical method: column – Vydac C18, 5 m, 2.1 x 250 mm; column temperature – 40°C; flow rate – 0.3 mL/min; solvent A – 0.01% aqueous TFA; solvent B – 70% CH3CN, 0.01% TFA; gradient – 0-20% B in 1 min., then 20-40% B in 20 min., then held at 100% B for 5 min.; when necessary the first two segments of the gradient were adjusted for compound lipophilicity; UV detection at 214 nm; injection volume – 5 µL, sample concentration – 0.5 mg/mL in 0.1% TFA. The purity of all analogues exceeded 95% (Table S4). Mass spectra were recorded on a MAT Finningan LCQ electrospray mass spectrometer. Synthesis of peptide alcohols 3 - 10. The linear precursors of the peptides were assembled on H-Arg(Pbf)-2ClTrt resin (commercially available) or H-D-Arg(Pbf)-2ClTrt resin using a Tribute Peptide Synthesizer (Protein Technologies Inc.). The Fmoc protecting group was removed with multiple 20% PIP/DMF treatments. The couplings were mediated by HBTU/NMM or DIC/HOBt for Cys. The Boc-protecting group was used at the N-terminus. The fully protected linear peptides were cleaved from resin with 30% HFIP/DCM. The C-terminal carboxylic group was reduced to the hydroxymethyl function by mixed anhydride method.48 The peptide alcohols were



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deprotected with the TFA/TIPS/H2O 96:2:2 cocktail and the linear peptides were cyclized in DMF under pseudo-high dilution conditions49 using HBTU/NMM coupling method. The cyclization reaction mixture was diluted with 10 volumes of 0.1% aqueous TFA and loaded onto HPLC column. The peptides were purified in a TFA based buffer and eluted from the column with a gradient of acetonitrile. Where necessary, a second purification in a TFA based buffer was performed. Synthesis of peptide secondary amides 11-18 and 20 - 37. FMPB AM resin (EMD Milipore, cat # 855028) was suspended in DCM/TMOF 1:1 mixture. An appropriate primary amine and solid sodium triacetoxyborohydride were subsequently added to the suspension. The resins were shaken overnight and washed with MeOH and DMF. The desired sequences were assembled on the Tribute synthesizer. The crude peptide amides were cleaved with the TFA/TIPS/H2O 96:2:2 cocktail and cyclized and purified as described above. Synthesis of Compound 19. A detailed protocol to prepare the compound has been recently published by us.56 The protected linear peptide was assembled manually starting from 7.8 g (6.9 mmol) of H-Pro-2ClTrt AM resin (EMD Milipore, cat # 856057, 0.88 mmol/g) using single, DIC/HOBt mediated couplings in DMF with a 3-fold excess of activated Fmoc-protected amino acids. The following amino acid derivatives were used to assemble the resin-bound peptide: Fmoc-Cys((CH2)3COOtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Thi-OH and BocCpa-OH. Removal of the Fmoc protecting group was achieved with a single 30 min. wash of the peptide resin with 20% piperidine in DMF. The fully protected peptide was cleaved with the DCM/HFIP 7:3 (v/v) cocktail (2 x 1 h, 30 mL each). The cleavage cocktails were pooled, the solvents were evaporated and the crude peptide was precipitated with ethyl ether, filtered and dried in vacuo. 5.79 g (4.63 mmol, 67 %) of the product was obtained.


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H-D-Arg-NEt2 x 2TFA. 2.81 g (5.4 mmol) of Boc-D-Arg(Pbf)-OH (Chem Impex, cat # 05282), 1.95 mL (11.2 mmol) of DIPEA and 2.13 g (5.6 mmol) of HBTU were dissolved in 10 mL DMF and 0.62 mL (6 mmol) of diethylamine was subsequently added to the solution. No substrate was detected by analytical HPLC after 5 min. The reaction mixture was poured into 500 mL of water and the precipitate was separated by centrifugation/decantation and dried in vacuo. The residue was treated with 20 mL TFA/TIPS/H2O (96/2/2, v/v/v) for 1 h and the solvents were evaporated. The residue was treated with diethyl ether, decanted and dried. 1.65 g (3.6 mmol, 67%) of semisolid derivative was obtained which was used in the subsequent step without purification. Boc-Cpa-Thi-Val-Asn(Trt)-Cys((CH2)3-COOtBu)-Pro-D-Arg-NEt2. 2.3 g (c.a. 1.86 mmol) of the linear protected peptide and 0.76 g (2 mmol) of HBTU were dissolved in 10 mL DMF containing 1.46 mL (8.4 mmol) DIPEA. 0.93 g (2.05 mmol) of H-D-Arg(Pbf)-OH x 2TFA in 1 mL DMF was subsequently added to the reaction mixture. No substrate protected peptide was detected after 5 min by HPLC. The product was precipitated with 1 L of water, filtered off and dried in vacuo. 2.6 g (1.78 mmol, 96%) of crude protected linear peptide was obtained. c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-D-Arg-NEt2, 19. The fully protected peptide was treated with 20 mL TFA/TIPS/H2O (96/2/2, v/v/v) for 1 h and the solvents were evaporated. The unprotected linear peptide was precipitated with ethyl ether and lyophilized. The entire amount of the linear peptide (1.82 g, 1.55 mmol) was dissolved in 50 mL of DMF. A solution of 0.59 g (c.a. 1.55 mmol) HBTU in 10 mL of DMF was also prepared. The peptide solution and the activator solution were added interchangeably to 50 mL of vigorously stirred DMF containing 200 uL of DIPEA in 10 portions of 5 mL and 1 mL, respectively. The pH was maintained at 9-10 with the addition of neat DIPEA. No substrate peak was detected by HPLC after the last portions



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of the activator and peptide solutions have been added. The reaction mixture was diluted with 0.1% AcOH to 1 L. The obtained solution was loaded directly onto an HPLC prep column and purified as described above. The fractions with a purity exceeding 93%, determined by reversephase analytical HPLC, were pooled, diluted with 2 volumes of water and reloaded onto the column. The column was washed with 5 volumes of 0.1M AcONH4 and one volume of 1% AcOH. The compound was subsequently eluted with a fast gradient of acetonitrile in 1% AcOH buffer to provide acetate salt. The fractions exceeding 95% purity were pooled and lyophilized. 703.1 mg (0.60 mmol, 22% overall based on 89.6% peptide content) of white peptide powder was obtained (see Supplemental Info, Table S4 for physicochemical properties). Synthesis of agmatine peptides 39, 41 - 43. The linear precursors of the peptides were assembled on H-Pro-2ClTrt resin (commercially available, 39, 41) or H-Thz-2ClTrt resin (42, 43) using a Tribute Peptide Synthesizer using synthetic protocols described above. The fully protected linear peptides were cleaved from resin with 30% HFIP/DCM. The fully protected peptide was coupled with agmatine (see Supplemental info for synthesis of compound 38). The protected linear analogues were deprotected, cyclized and purified as described above. The synthesis of compound 40 and additional synthetic protocols can be found in Supporting Info.

Biological methods In vitro receptor assays. The agonist activity and potency of compounds at the human and rat vasopressin V2R receptor (hV2R) were determined in a transcriptional reporter gene assay (RGA) by transiently cotransfecting recombinant human or rat V2 receptor expression DNA construct into a human embryonic kidney (HEK-293) cell line with a reporter DNA construct containing a luciferase gene under the control of cAMP responsive promoter elements. Two days following 32 ACS Paragon Plus Environment

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transfection, cells were treated with appropriate concentrations of peptides, incubated for 5 h, followed by lysis of cells in the presence of luciferin and the total luminescence was measured. To determine receptor selectivity, compounds were also tested in luciferase-based transcriptional reporter assays in HEK293 cells expressing the human V1aR and human V1bR. Assays for human oxytocin OTR activity were performed in CHO-K1 cells stably expressing human OT receptor and transiently transfected with the NFAT-luciferase reporter construct. Compound 1 was used as an internal control for the V2R assays, AVP was used as an internal control for the vasopressin V1aR and V1bR assays, and carbetocin (carba-1[Tyr(Me)2]dOT) was used as an internal control for the OTR assays. The assays were standardized by including these controls in every experiment. In all assays compounds were tested in at least three independent experiments performed in duplicates. Dose-response curves were analyzed using a one-site, four parameter model from Xlfit (IDBS) and used to estimate EC50 and efficacy values. Agonist potencies determined from multiple independent experiments are reported by first calculating pEC50 values from EC50 (pEC50 = -log(EC50 in M)) for each individual value, and then determining the 95% CI of pEC50. These values are then transformed back to linear scale. This is based on the assumption that the data follows a Gaussian distribution, that the values are randomly sampled, and the sample assumes the same distribution as the theoretical full statistical population. Agonist efficacy is presented as the arithmetic means in percentage of internal control efficacy. The final efficacy is reported as arithmetic mean ± 95% CI. Selectivity values are given as ratios of the EC50 values at the receptor of interest to the corresponding EC50 values at the V2R. PK and PD studies. Animals. All procedures involving the use of animals were approved by the Ferring Research Institute or Contract Research Organization Institutional Animal Care and



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Use Committee and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council. The single-dose pharmacokinetic profile and antidiuretic activity of V2 analogues were investigated following iv administration in male Sprague-Dawley rats (230 – 380 g, age commensurate with body weight) at Ferring Research Institute. Naïve rats or rats with chronic jugular vein and carotid artery catheters inserted surgically were obtained from Harlan Laboratories Inc. (Indianapolis USA). The rats were given free access to food (18% protein Rodent Diet, Harlan Teklad, Madison USA) and water. They were housed in a conventional animal facility in individually ventilated caging (LabProducts Inc., Seaford USA) with appropriate air flow under controlled environmental conditions (2022ºC, 12 h light/dark cycle). In-life portion of the monkey, dog and minipig single-dose pharmacokinetic studies were conducted at Sinclair Research Centre (Auxvasse MO USA). Cynomolgus monkey (male, average body weight 5 kg, age commensurate with body weight), Beagle dog (male, average body weight 9 kg, age 15 months) and Yucatan minipig (castrated male, average body weight 70 kg, age 3.5 years) were used in the studies.

In-life experimental protocol for non-renal CL determination in nephrectomised rats. These experiments were conducted in terminally anesthetized rats. On each experimental day, animals were lightly anesthetized with isoflurane and then injected with the long-acting aesthetic thiobutabarbital. Once under anesthesia, each animal was placed on a heating pad and implanted with jugular vein and carotid artery catheters, for compound administration and blood sampling respectively, and a tracheostomy tube for assisted respiration. Bilateral kidney occlusion was performed by making an abdominal incision and ligating the renal blood vessels and ureter with suture. Sham surgery was performed using identical procedures including kidney manipulation


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and placement of sutures without ligation. Following the surgical procedures, animals were connected to ventilators (Harvard Apparatus) through their tracheostomy tubes and allowed to stabilize for 30 minutes before compound administration. PK Studies - Dosing and sampling. The analogues were dissolved in 5% mannitol in water. Each rat was given a single dose of 0.1 mg/kg of each test compound in cassette mode through the jugular vein.57 Cassettes were constituted of 3 to 6 V2 analogues with each compound at a concentration of 0.1 mg/mL. Blood samples (250 μL) were collected from the carotid artery catheter at nominal times of either 2, 6, 10, 15, 20, 30, 45, 60, 90 and 120 min or 2, 6, 10, 15, 20, 40, 60, 90, 135 and 180 min after administration into pre-chilled tubes containing K2EDTA as anticoagulant. Blood that was drawn from the animal was replaced with an equal volume of saline. The samples were centrifuged at 4⁰C and plasma was separated. All samples were immediately frozen on dry ice and stored at -50ºC until further analysis. . Similar formulation and intravenous dosing procedures were used for monkey, dog and minipig studies. Monkeys and dogs were dosed intravenously at 0.05 mg/kg, whereas minipigs were dosed at 0.01 mg/kg. 11 blood samples were taken between 3 min and 180 min postdose. Bioanalysis. The concentrations of compounds in rat monkey, dog and minipig plasma were determined using a liquid chromatography tandem mass spectrometry (LC/MS/MS) method. The dynamic range of the assays was 1-1000 ng/mL. Standard and internal standard solutions were prepared in 0.1% TFA in 100% acetonitrile. Typically, 50 μL of rat plasma sample was mixed with 30 μL of the internal standard and 200 μl of the crashing solution (0.1% TFA in 100% acetonitrile). After shaking for 2 min and centrifugation at 5700 g for 45 min, 250 μL of supernatant was concentrated by evaporation under nitrogen at 35ºC for 15 min. Samples were then reconstituted with 100 μL of 0.01% TFA in 14% acetonitrile. Reconstituted samples were



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centrifuged again for 30 min at 5700 g. 40 μL of supernatant was injected into a Phenomenex Luna 3 µm 100Å C18(2) 50 x 2.0 mm HPLC column (Phenomenex, Torrance USA) coupled to a Shimadzu Nexera LC (Shimadzu, Kyoto Japan) system. The analytes were eluted using a gradient of solvent B (solvent A - 0.01% TFA and 1% formic acid in water; solvent B - 0.01% TFA, 1% formic acid and 70% acetonitrile in water) at a flow rate of 0.6 mL/min and detected using an API-5500 triple quadrupole mass spectrometer (Applied Biosystems, Ontario Canada) in the positive electrospray ionization mode. Analyte concentrations were calculated by linear regression analysis using the peak area ratio of analyte to the internal standard on the Applied Biosystems Analyst software version 1.4.2. PK data analysis. PK parameters were calculated using a noncompartmental (NCA) method with Phoenix WinNonlin 6.1 (Certara, St. Louis USA). The area under the plasma concentrationtime curve to infinity (AUC∞) was calculated by combining the area under the curve to the last time point (AUC0-t) with the extrapolated AUC value of the terminal phase. Body weight normalized CL values (mL/min/kg) were calculated as dose divided by AUC(0-∞) and rat body weight. The percentage of non-renal clearance (% CLnr) was determined by comparing whole body compound clearance in functionally nephrectomized animals to sham-operated animals: 𝐶𝐿𝑛𝑟(%) =

𝐶𝐿𝑛𝑒𝑝ℎ𝑟𝑒𝑐𝑡𝑜𝑚𝑖𝑧𝑒𝑑 𝐶𝐿𝑠ℎ𝑎𝑚 ― 𝑜𝑝𝑒𝑟𝑎𝑡𝑒𝑑

× 100%

Determination of rat plasma protein binding. Rat plasma protein binding (PPB) analysis was performed by an ultracentrifugation (UC) method. Briefly, 5 µL of a 20 - 100 μg/mL solution of the V2 analogue in 0.01% TFA in 35% acetonitrile was added to a 1 mL Sorvall polycarbonate thick-walled UC tube (Fisher Scientific, product number 45237) containing 495 μL of rat plasma (Bioreclamation, Westbury NY USA) to a final peptide concentration of 200 36 ACS Paragon Plus Environment

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1000 ng/mL. The peptide plasma mixture was incubated at 37 °C for 15 min in a Sorvall S140AT 35° fixed angle rotor, and then spun at 120,000 rpm (773,000 xg) for 30 min at 37 °C in a Sorvall MX150 micro-ultracentrifuge (Asheville NC, USA). After centrifugation, 50 µL sample in the middle layer of the UC tube was collected at the 300 μL volume meniscus level with a 250 µL pipettor tip. The compound concentrations in the collected sample was quantified immediately by the LC/MS/MS method described in the Experimental section. Each compound was assayed in quadruplicate. The percentage of binding was calculated as: 𝑃𝑃𝐵(%) =

𝐶𝑖 ― 𝐶𝑚 × 100 𝐶𝑖

Where Ci is the initial concentration of peptide and Cm is the concentration of peptide remaining in the middle layer after ultracentrifugation.

Rat antidiuretic activity assay. Antidiuretic activity of selected V2 analogues was assessed in rats by measuring urine output during and after administration of compound. Compound formulation and dose calculation. Compounds were formulated in 2.5% (w/v) rat or bovine serum albumin in nanopure water. On the day prior to each experiment, compounds were formulated at the concentrations to be used in the experiment and all tubing, syringes, and vials to be used for compound formulation and animal dosing were pre-equilibrated overnight with compound solution in attempt to mitigate potential compound non-specific binding. On the day of study, the compound solution that was used for pre-equilibration was discarded and fresh compound was weighed into the pre-equilibrated vials. Whole body clearance (CL) values determined from rat PK studies (preliminary PK, cassette dosing) and PK simulations (Phoenix WinNonlin 6.1, Certara, St. Louis, USA) were used to calculate the iv infusion rates (with



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optional loading dose during the first 20 min.) to achieve targeted steady state concentrations (Css) ranging from 0.3 to 100 pM within 15-30 min of infusion. In-life experimental protocol. On each experimental day, animals were weighed, assigned to treatment groups to receive test compound or 1 at one target Css, or vehicle, and placed into metabolic cages for urine collection with free access to water, but no food. Average total vehicle volume was 1.2 mL per rat over 3 hours. Rat receiving compounds were infused with an identical volume of vehicle. Under these experimental conditions, rats are expected to be euvolemic as the volume used for compound administration was relatively low compared to antidiuresis studies that use acute large volume water-loading to induce hypervolemia. Each metabolic cage was set up for continuous measurement of spontaneous urine output via force transducers attached to the urine collection vials to monitor and record cumulative urine weight. Catheters were attached to a swivel/tether. Compounds or vehicle were administered by IV infusion using a syringe infusion pump for the first 180 min of urine output measurements. At 180 min, the compound infusion was stopped and the urine output was measured for an additional 300 min. At the end of the experiment, animals were returned to their home cages. Animals were used in up to three experiments separated by at least one week. Data Analysis. Cumulative urine output time courses (time points at 10 min intervals from 0 to 8 h) collected over multiple experiments were compiled; mean, SEM, and N were determined for each treatment group and time point. Dose-response analysis was performed on the compiled urine output data at the 180 min time point (end of infusion) in each treatment group. Compound potency was expressed as the ECp50 value, the effective plasma concentration of compound (targeted Css in pM) causing half-maximal suppression of urine output, calculated using a 3 parameter concentration response logistic model.


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ASSOCIATED CONTENT Supporting Information. In vitro, PK, PPB data with statistical parameters, and physiochemical properties of analogues 1 – 43 as well as the description of additional synthetic methods are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Kazimierz Wiśniewski: 0000-0003-0002-5411 Notes The authors declare no competing financial interest ACKNOWLEDGMENT The authors thank Monica Mares and Ying Zhang for their excellent technical assistance and Jolene Lau for providing relevant intelligence information. We also wish to thank Leslie Callejas-Dominguez for her assistance in implementing the rat antidiuretic activity/duration of action assay and Denise Riedl for auditing the data and critical reading of the manuscript. ABBREVIATIONS Aaa, any amino acid residue; Agm, agmatine, N-(4-aminobutyl)guanidine; Bua, butyric acid; BW, body weight; CL, systemic clearance; Cpa, β-(4-chlorophenyl)alanine; cPr, cyclopropyl;



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DIC, N,N’-diisopropylcarbodiimide; DIPEA, N,N-diisopropyl-N-ethylamine; EtThi, 2-(2thienyl)ethyl; Fpa, β-(4-fluorophenyl)alanine; GFR, glomelural filtration rate; HBTU, 2-(1Hbezotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate;

HFIP,

1,1,1,3,3,3-

hexafluoro-2-propanol; HOBt, 1-hydroxybenzotriazole, hV1aR, human vasopressin 1a receptor; hV1bR, human vasopressin 1b receptor; hV2R, human vasopressin 2 receptor; Hyp, transhydroxyproline, (2S,4R) 4-hydroxyproline; i-Bu, isobutyl; MeOH, methanol; NMM, Nmethylmorpholine, 4-methylmorpholine; PPB, plasma protein binding; RGA, reporter gene assay; rV2R, rat vasopressin 2 receptor; Thi, β-(2-thienyl)alanine; Thz, thiaproline, (S)thiazolidine-4carboxylic acid; TMOF, trimethyl orthoformate REFERENCES (1)

Liu, J.; Sharma, N.; Zheng, W.; Ji, H.; Tam, H.; Wu, X.; Manigrasso, M. B.; Sandberg,

K.; Verbalis, J. G. Sex Differences in Vasopressin V(2) Receptor Expression and VasopressinInduced Antidiuresis. Am. J. Physiol. Renal Physiol. 2011, 300, F433-440. (2)

Zaoral, M.; Kolc, J.; Sorm, F. Amino Acids and Peptides. LXXI. Synthesis of 1-

Deamino-8-D-γ-Aminobutyrine Vasopressin, 1-Deamino-8-D-Lysine Vasopressin, and 1Deamino-8-D-Arginine Vasopressin. Collect. Czech. Chem. Commun. 1967, 32, 1250-1257. (3)

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Mannucci, P. M. Treatment of Von Willebrand's Disease. N. Engl. J. Med. 2004, 351,

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Fenske, W.; Hoorn, E. J.; Ichai, C.; Joannidis, M.; Soupart, A.; Zietse, R.; Haller, M.; van der Veer, S.; Van Biesen, W.; Nagler, E. Clinical Practice Guideline on Diagnosis and Treatment of Hyponatraemia. Intensive Care Med. 2014, 40, 320-331. (10) Vande Walle, J.; Stockner, M.; Raes, A.; Norgaard, J. P. Desmopressin 30 Years in Clinical Use: A Safety Review. Curr. Drug Saf. 2007, 2, 232-238. (11) Rittig, S.; Knudsen, U. B.; Norgaard, J. P.; Pedersen, E. B.; Djurhuus, J. C. Abnormal Diurnal Rhythm of Plasma Vasopressin and Urinary Output in Patients with Enuresis. Am. J. Physiol. 1989, 256, F664-671. (12) Vande Walle, J. G.; Bogaert, G. A.; Mattsson, S.; Schurmans, T.; Hoebeke, P.; Deboe, V.; Norgaard, J. P. A New Fast-Melting Oral Formulation of Desmopressin: A 41 ACS Paragon Plus Environment


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Pharmacodynamic Study in Children with Primary Nocturnal Enuresis. BJU Int. 2006, 97, 603609. (13) Lucchini, B.; Simonetti, G. D.; Ceschi, A.; Lava, S. A.; Fare, P. B.; Bianchetti, M. G. Severe Signs of Hyponatremia Secondary to Desmopressin Treatment for Enuresis: A Systematic Review. J. Pediatr. Urol. 2013, 9, 1049-1053. (14) Bosch, J. L.; Weiss, J. P. The Prevalence and Causes of Nocturia. J. Urol. 2013, 189, S86-92. (15) Hofmeester, I.; Kollen, B. J.; Steffens, M. G.; Bosch, J. L.; Drake, M. J.; Weiss, J. P.; Blanker, M. H. The Association between Nocturia and Nocturnal Polyuria in Clinical and Epidemiological Studies: A Systematic Review and Meta-Analyses. J. Urol. 2014, 191, 10281033. (16) Van Kerrebroeck, P. Nocturia: Current Status and Future Perspectives. Curr. Opin. Obstet. Gynecol. 2011, 23, 376-385. (17) Sand, P. K.; Dmochowski, R. R.; Reddy, J.; van der Meulen, E. A. Efficacy and Safety of Low Dose Desmopressin Orally Disintegrating Tablet in Women with Nocturia: Results of a Multicenter, Randomized, Double-Blind, Placebo Controlled, Parallel Group Study. J. Urol. 2013, 190, 958-964. (18) Weiss, J. P.; Herschorn, S.; Albei, C. D.; van der Meulen, E. A. Efficacy and Safety of Low Dose Desmopressin Orally Disintegrating Tablet in Men with Nocturia: Results of a Multicenter, Randomized, Double-Blind, Placebo Controlled, Parallel Group Study. J. Urol. 2013, 190, 965-972.


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(19) Agerso, H.; Seiding Larsen, L.; Riis, A.; Lovgren, U.; Karlsson, M. O.; Senderovitz, T. Pharmacokinetics and Renal Excretion of Desmopressin after Intravenous Administration to Healthy Subjects and Renally Impaired Patients. Br. J. Clin. Pharmacol. 2004, 58, 352-358. (20) Callreus, T.; Ekman, E.; Andersen, M. Hyponatremia in Elderly Patients Treated with Desmopressin for Nocturia: A Review of a Case Series. Eur. J. Clin. Pharmacol. 2005, 61, 281284. (21) Fjellestad-Paulsen, A.; Hoglund, P.; Lundin, S.; Paulsen, O. Pharmacokinetics of 1Deamino-8-D-Arginine Vasopressin after Various Routes of Administration in Healthy Volunteers. Clin. Endocrinol. (Oxf) 1993, 38, 177-182. (22) Odeberg, J. M.; Callreus, T.; Lundin, S.; Roth, E. B.; Hoglund, P. A Pharmacokinetic and Pharmacodynamic Study of Desmopressin: Evaluating Sex Differences and the Effect of PreTreatment with Piroxicam, and Further Validation of an Indirect Response Model. J. Pharm. Pharmacol. 2004, 56, 1389-1398. (23) Neveus, T.; Lackgren, G.; Tuvemo, T.; Stenberg, A. Osmoregulation and Desmopressin Pharmacokinetics in Enuretic Children. Pediatrics 1999, 103, 65-70. (24) Ruzicka, H.; Bjorkman, S.; Lethagen, S.; Sterner, G. Pharmacokinetics and Antidiuretic Effect of High-Dose Desmopressin in Patients with Chronic Renal Failure. Pharmacol. Toxicol. 2003, 92, 137-142. (25) Rembratt, A.; Graugaard-Jensen, C.; Senderovitz, T.; Norgaard, J. P.; Djurhuus, J. C. Pharmacokinetics and Pharmacodynamics of Desmopressin Administered Orally Versus



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Intravenously at Daytime Versus Night-Time in Healthy Men Aged 55-70 Years. Eur. J. Clin. Pharmacol. 2004, 60, 397-402. (26) Caggiano, T. J. WAY-VNA-932. Drugs Future 2002, 27, 248-253. (27) Nakamura, S.; Yamamura, Y.; Itoh, S.; Hirano, T.; Tsujimae, K.; Aoyama, M.; Kondo, K.; Ogawa, H.; Shinohara, T.; Kan, K.; Tanada, Y.; Teramoto, S.; Sumida, T.; Nakayama, S.; Sekiguchi, K.; Kambe, T.; Tsujimoto, G.; Mori, T.; Tominaga, M. Characterization of a Novel Nonpeptide Vasopressin V(2)-Agonist, OPC-51803, in Cells Transfected Human Vasopressin Receptor Subtypes. Br. J. Pharmacol. 2000, 129, 1700-1706. (28) Yea, C. M.; Allan, C. E.; Ashworth, D. M.; Barnett, J.; Baxter, A. J.; Broadbridge, J. D.; Franklin, R. J.; Hampton, S. L.; Hudson, P.; Horton, J. A.; Jenkins, P. D.; Penson, A. M.; Pitt, G. R.; Riviere, P.; Robson, P. A.; Rooker, D. P.; Semple, G.; Sheppard, A.; Haigh, R. M.; Roe, M. B. New Benzylureas as a Novel Series of Potent, Nonpeptidic Vasopressin V2 Receptor Agonists. J. Med. Chem. 2008, 51, 8124-8134. (29) Koshio, H.; Tsukamoto, I.; Kakefuda, A.; Akamatsu, S.; Saitoh, C. Preparation of 4,4Difluoro-1,2,3,4-Tetrahydro-1(5H)-Benzazepine Derivatives for Treatment of Night Pollakisuria and Diabetes Insipidus. WO2004096775A1, 2004. (30) Katayama, R.; Yamaguchi, N.; Yamashita, T.; Watanabe, S.; Satoh, H.; Yamagishi, N.; Furuhama, K. Calculation of Glomerular Filtration Rate in Conscious Rats by the Use of a Bolus Injection of Iodixanol and a Single Blood Sample. J. Pharmacol. Toxicol. Methods 2010, 61, 5964.


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(31) Manning, M.; Stoev, S.; Chini, B.; Durroux, T.; Mouillac, B.; Guillon, G. Peptide and Non-Peptide Agonists and Antagonists for the Vasopressin and Oxytocin V1a, V1b, V2 and OT Receptors: Research Tools and Potential Therapeutic Agents. Prog. Brain Res. 2008, 170, 473512. (32) Manning, M.; Misicka, A.; Olma, A.; Bankowski, K.; Stoev, S.; Chini, B.; Durroux, T.; Mouillac, B.; Corbani, M.; Guillon, G. Oxytocin and Vasopressin Agonists and Antagonists as Research Tools and Potential Therapeutics. J. Neuroendocrinol. 2012, 24, 609-628. (33) Sawyer, W. H.; Acosta, M.; Balaspiri, L.; Judd, J.; Manning, M. Structural Changes in the Arginine Vasopressin Molecule That Enhance Antidiuretic Activity and Specificity. Endocrinology 1974, 94, 1106-1115. (34) Kowalczyk, W.; Sobolewski, D.; Prahl, A.; Derdowska, I.; Borovickova, L.; Slaninova, J.; Lammek, B. The Effects of N-Terminal Part Modification of Arginine Vasopressin Analogues with 2-Aminoindane-2-Carboxylic Acid: A Highly Potent V2 Agonist. J. Med. Chem. 2007, 50, 2926-2929. (35) Kwiatkowska, A.; Sobolewski, D.; Prahl, A.; Borovickova, L.; Slaninova, J.; Lammek, B. Arginine Vasopressin and Its Analogues - The Influence of Position 2 Modification with 3,3Diphenylalanine Enantiomers. Highly Potent V2 Agonists. Eur. J. Med. Chem. 2009, 44, 28622867. (36) Ben Mimoun, M.; Derick, S.; Andres, M.; Guillon, G.; Wo, N. C.; Chan, W. Y.; Stoev, S.; Cheng, L.; Manning, M. Vasopressin V2 Agonists: Affinities for Human and Rat V2 and V1a Receptors Reveal Surprising Species Differences. In Peptides 2000, Proceedings of the 26th



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European Peptide Symposium, Montpelier, France, September 10-15, 2000, Martinez, J.; Fehrentz, J.-A., Eds. Editions EDK: 2001; pp 589-590. (37) Manning, M.; Olma, A.; Klis, W.; Kolodziejczyk, A.; Nawrocka, E.; Misicka, A.; Seto, J.; Sawyer, W. H. Carboxy Terminus of Vasopressin Required for Activity but Not Binding. Nature 1984, 308, 652-653. (38) Manning, M.; Przybylski, J. P.; Olma, A.; Klis, W. A.; Kruszynski, M.; Wo, N. C.; Pelton, G. H.; Sawyer, W. H. No Requirements of Cyclic Conformation of Antagonists in Binding to Vasopressin Receptors. Nature 1987, 329, 839-840. (39) Manning, M.; Cheng, L. L.; Misika, A.; Olma, A.; Klis, W. A.; Bankowski, K.; Nawrocka, E.; Kruszynski, M.; Kolodziejczyk, A.; Sawyer, W. H. C-Terminal Deglycine and Deglycinamide Modifications of Arginine Vasopressin (AVP) Agonists and Antagonists. A Reevaluation. Pept. Chem. 1988, 585-590. (40) Manning, M.; Misicka, A.; Olma, A.; Klis, W. A.; Bankowski, K.; Nawrocka, E.; Kruszynski, M.; Kolodziejczyk, A.; Cheng, L. L.; Seto, J.; Wo, N. C.; Sawyer, W. H. CTerminal Deletions in Agonistic and Antagonistic Analogues of Vasopressin That Improve Their Specificities for Antidiuretic (V2) and Vasopressor (V1) Receptors. J. Med. Chem. 1987, 30, 2245-2252. (41) Mahmood, I.; Martinez, M.; Hunter, R. P. Interspecies Allometric Scaling. Part I: Prediction of Clearance in Large Animals. J. Vet. Pharmacol. Ther. 2006, 29, 415-423. (42) Mahmood, I. Application of Allometric Principles for the Prediction of Pharmacokinetics in Human and Veterinary Drug Development. Adv. Drug Deliv. Rev. 2007, 59, 1177-1192.


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(50) Fujii, N.; Otaka, A.; Sugiyama, N.; Hatano, M.; Yajima, H. Studies on Peptides. CLV. Evaluation of Trimethylsilyl Bromide as a Hard-Acid Deprotecting Reagent in Peptide Synthesis. Chem. Pharm. Bull. (Tokyo) 1987, 35, 3880-3883. (51) Rodrigo, J.; Pena, A.; Murat, B.; Trueba, M.; Durroux, T.; Guillon, G.; Rognan, D. Mapping the Binding Site of Arginine Vasopressin to V1a and V1b Vasopressin Receptors. Mol. Endocrinol. 2007, 21, 512-523. (52) Singer, M. A.; Morton, A. R. Mouse to Elephant: Biological Scaling and Kt/V. Am. J. Kidney Dis. 2000, 35, 306-309. (53) Verbalis, J. G. Disorders of Body Water Homeostasis. Best Pract. Res. Clin. Endocrinol. Metab. 2003, 17, 471-503. (54) Juul, K. V.; Erichsen, L.; Robertson, G. L. Temporal Delays and Individual Variation in Antidiuretic Response to Desmopressin. Am. J. Physiol. Renal Physiol. 2012, 304, F268-278. (55) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. 2-Chlorotrityl Chloride Resin. Studies on Anchoring of Fmoc-Amino Acids and Peptide Cleavage. Int. J. Pept. Protein Res. 1991, 37, 513-520. (56) Wisniewski, K.; Schteingart, C.; Riviere, P. Preparation of Cyclic Peptide Vasopressin-2 Receptor Agonists. WO2015013690A1, 2015. (57) Frick, L. W.; Adkison, K. K.; Wells-Knecht, K. J.; Woollard, P.; Higton, D. M. Cassette Dosing: Rapid in Vivo Assessment of Pharmacokinetics. Pharm. Sci. Technol. Today 1998, 1, 12-18.


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TABLE OF CONTENTS GRAPHIC



Table of Contents Graphic 254x190mm (96 x 96 DPI)


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Figure 4. Allometric scaling for selected compounds. Panel A (compound 31, carba-6), B (11, disulfide), C (19, carba-1) and D (38, carba-1). The CL values used to construct the graphs are listed in Supplemental Info (Tables S1, S2). The CL values are represented by blue diamonds (rat), red dots (Cyno), orange squares (dog) and green triangles (pig). 3 animals per compound were used with the exception of rat experiments where 4 animals were used. Additional allometric scaling plots are shown in Fig. S1. 254x190mm (96 x 96 DPI)



Figure 5. Comparison of in vivo antidiuretic potency and maximal effect of 1 and 19 (left graph) and 1 and 38 (right graph) in normal euvolemic rats assessed by measuring weight of cumulative urine output at 3h of intravenous infusion. Shown are mean ± SEM for urine output in N = 200 vehicle-treated animals and 15 20 compound-treated animals at each target Css (error bars that are not visible are encompassed within the symbol). Fitted parameters for dose-response are summarized in Table 7. 254x190mm (96 x 96 DPI)


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Figure 6. Rat antidiuretic time course at maximally effective target Css. Time courses of 19 (left graph) and 38 (right graph) (30 and 100 pM) compared to 1. Peptides were infused iv through 180 min. at doses to rapidly achieve and maintain the indicated target Css and cumulative urine weight was measured. At 180 min., peptide infusion was stopped and urine output was measured for an additional 300 min. Shown are mean urine output in N = 200 vehicle-treated animals and 16 - 20 compound-treated animals. Error bars (only shown at 60 minute intervals for clarity) indicate ± SEM in each treatment group. 254x190mm (96 x 96 DPI)


Discovery of Potent, Selective and Short-Acting Peptidic V2 Receptor

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