European Journal of Pharmacology, 204 ( 1991) 179-185 © 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50

179

EJP 52102

Human growth hormone-releasing hormone analogues with much improved in vitro growth hormone-releasing potencies in rat pituitary cells David H. Coy, Simon J. H o c a r t and William A. M u r p h y Peptide Research Laboratories, Department of Medicine, Tulane University Medical Center, New Orleans, L.A. 70112, U.S.A. Received 10 May 1991, accepted 6 August 1991

Enhancement of the amphiphilic a-helical properties of the central and C-terminal regions of growth hormone-releasing hormone (GRH) by substitution with helix-favouring amino acids, particularly Ala, can result in significant improvements in GH-releasing potencies using monolayer cultures of rat pituitary cells, a system which reflects analogue receptor affinity rather than effects of structural modifications on pharmacokinetic properties. For instance, previously reported, helix-enhanced [AlalS]GRH-(1-29)NH2 was presently 5 times more potent than [GlyIS]GRH-(I-29)NH2 in this assay. The extent and importance of a-helical character further towards the N-terminus is less clear since Chou-Fasman probability calculations indicate also the possibility of fl-bend formation in the 6-10 region. However, replacement of Asn 8 with Ala resulted in a 4-fold improvement in potency and when this was combined with Ala 15 to give [AlaS'IS]GRH-(1-29)NH2 a 15-fold increase in potency was achieved and combination of D-Ala 2, Ala s and Ala ~5 gave a 27-fold increase indicating that the effects of all of these modifications were additive. Computer analysis furthermore revealed that substitution of Ala for Ser in position 9 should also increase a-helix probability from 0.93 to 1.05. [D-Ala2,AlaS'~aS]GRH-(l-29)NH2 was 49 times more potent than GRH itself making it by far the most potent analogue thus far reported in an in vitro assay system. The Ala s and Ala9 substitutions were also effective in improving the inhibitory potency of a GRH receptor antagonist, [D-Arg2,Leu27]GRH-(1-29)NH,. [DArg2,AlaS'lS]GRH-(1-29)NH2 and [D-ArgZ,AlaS'~'IS]GRH-(I-29)NH2 displayed ICstI values of 5.9 × 10 -s and 1.7 × 10--s M, respectively, against GRH-stimulated GH release compared to an IC5o of 2.2 × 10 7 M for the unmodified control analogue and are thus commensurate with corresponding agonist analogue potency improvements. GRH (growth hormone-releasing hormone); GRH analogues; Pituitar3~cells (rat); (Agonists); (Antagonists)

1. Introduction Human growth hormone-releasing hormone (GRH) was originally isolated and characterized by Guiilemin et al. (1982) and Rivier et al. (1982) as 44 and 40 residue peptides, respectively, and found to be structurally closely related to the glucagon-secretin-VIP family of peptides. Virtually full biological activity resides in the first 29 residues (Rivier et al., 1982; Lance et al., 1984) and the 1-29-amidated structure has provided the basis for most structure-activity studies. These physicochemical and synthetic studies strongly suggest that the C-terminal two thirds of the G R H - ( 1 29)NH 2 chain (fig. 1) adopts a predominantly amphiphilic a-helical conformation both in solution and in its receptor bound state (Velicebili et al., 1986).

Correspondence to: D.H. Coy, Peptide Research Laboratories, Department of Medicine, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, LA 7011Z U.S.A. Tel. !.504.584 3584, fax 1.504.584 3586.

Indeed, multiple amino acid replacements can be readily made which completely retain or even slightly increase biological potency as long as their side-chains retain these global amphiphilic characteristics (Velicebili et ai., 1986; Tou et al., 1986). Chou-Fasman analysis (Chou and Fasman, 1974; Murphy et al., 1986), however, reveals that certain parts of the G R H peptide chain are not as structurally optimized as they could be in the favor of a-helix formation (see table 1). In particular, Giy in position 15 could be replaced by Ala to significantly improve predicted a-helicity and also biological potency by about a factor of 3 in vitro in the rat (Felix et al., 1987) However, the pret~rred conformation of the N-terminal decapeptide region of G R F is less clear. Substitution of D-Ala in position 2 of GRH-(1-29)NH 2 (Lance et al., 1984) gave around a 50-fold increase in GH releasing potency in the anesthetized rat and it was proposed that some folding was thus favored at the N-terminus of the chain since D-amino acids can frequently favor/3-bend formation. Subsequently, this potency level could not be achieved in humans (Grossman et al., 1984) or in vitro (Murphy

-tl

iD-Ala) I

t-NH 2 Ala-9 (093 ~

105)

Fig. 1. Stabilization of proposed amphiphilic a-helix conformation of GRH-(1-29)NH 2 by substitutions with Ala at the shaded positions and showing changes in calculated Chou-Fasman helix probabilities before and after.

and Coy, 1988) and it can be concluded that this and certain other N-terminal modifications, such as desamino-Tyr (Felix et al., 1988), result in large increases in in vivo potency by blocking dipeptidylpeptidase activity, now known to be a major mechanism for plasma GRH degradation (Frohman et al., 1986; Kubiak et al., 1989) in several species. We also showed by Chou-Fasman computer analysis that /3-bend formation around Asn 8 is more favorable than a-helicity (Murphy and Coy, 1988) (table 1) and the increased potency of a D-Asn 8 analogue both in vivo (Coy et al., 1985) and in vitro (table 2) seemed to support this conclusion. On the other hand, bridging of the position 8 (Asn) and 12 (Lys) side chains with retention of biological potency (Felix et al., 1988) suggested that a-helicity extended at least to position 8 since these side chains would then be predicted to be in close 3-dimensional proximity. We have now re-examined this situation by incorporating amino acids specifically into position 8 and 9 whose side chains should either decrease the tendency to fold by enhancing a-helicity in this region according to the Chou-Fasman probability constants or, conversely, promote folding via incorporation of D-amino acids with various side-chains or N-Me amino acids. Analogues with high potency have also been further enhanced using the previously discussed D-?da 2 and Ala ~5 replacements in an effort to obtain maximally potent peptides. The effects of some of the same substitutions on the in vitro potency of a competitive receptor GRH antagonist, [D-Arg2]GRH (Robberecht et al., 1985), were also examined in order to discern whether its inhibitory potency would be similarly affected.

scribed by Coy et al. (1985) on methylbenzhydrylamine resin (Advanced ChemTech, Louisville, KY) using an automatic ACT 200 peptide synthesizer (Advanced ChemTech, Louisville, KY). Briefly, the crude hydrogen fluoride-10% m-cresol-cleaved peptide amides were purified on a column (2.5 x 90 cm) of Sephadex G-50 which was eluted with 2 M acetic acid followed by preparative medium pressure chromatography on a column (1.5 x 45 cm) of Vydac C~8 silica (10-15/zm) which was eluted with linear gradients of acetonitrile in 0.1% trifluoroacetic acid (flow rate ca. 6 ml/min). Analogues were further purified by re-chromatography on the same column with slight modifications to the gradient conditions when necessary. Homogeneity of the peptides was assessed by thin layer chromatography and analytical reverse-phase high pressure liquid chromatography and purity was 97% or higher. Amino acid analysis of acid hydrolysates gave the expected amino acid ratios. The correct structures of some key analogues were further demonstrated by fast atom bombardment mass spectrometry whereupon each analogue gave good recovery of the molecular ion corresponding to its calculated molecular weight.

2.2. Chou-Fasman analysis Empirical probability calculations for potential ahelical,/3-sheet, and reverse turn content of GRH-(129)NH 2 and analogues were carried out using an inhouse computer program employing the protein-derived data base compiled by Argos et al. (1982).

2.3. Pituitary cell dispersion 2. Materials and methods

2.1. Preparation of peptides Solid-phase syntheses of GRH peptide amides were carried out essentially by the standard methods de-

Anterior pituitaries from adult male Charles River rats weighing 200-250 g and housed under controlled conditions (lights on from 0:50 to 19:00 h), were dispersed using aseptic technique by a previously described trypsin/DNase method (Murphy and Coy, 1988; Heiman et al., 1985).

181

2.4. Pituitary cell culture The dispersed cells were diluted with sterile-filtered Dulbecco's modified Eagle medium (MEM) (Gibco Laboratories, Grand Island, NY (GIBCO)), which was supplemented with 2.5% fetal calf serum (GIBCO), 3% horse serum (GIBCO), 10% fresh rat serum (stored on ice for no longer than 1 h) from the pituitary donors, 1% MEM nonessential amino acids (GIBCO), gentamycin (10 ng/ml; Sigma) and nystatin (10000 U / m l ; GIBCO). The cells were counted with a hemacytometer (approximately 2000000 cells per pituitary) and randomly plated at a density of 200000 cells per well (Co-star cluster 24; Rochester Scientific Co., Rochester, NY). The plated cells were maintained in the above Dulbecco's medium in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C for 96 h.

2.5. In vitro pituitary incubation In preparation for a hormone challenge, the cells were washed 3 × with medium 199 (GIBCO) to remove old medium and floating cells. Each dose of secretagogue (diluted in siliconized test tubes) was tested in quadruplicate wells in a total volume of 1 ml medium 199 containing 1% BSA (fraction V; Sigma Chemical Co., St. Louis, MO). Cells were pulsed in the presence of 0.1 nM somatostatin (Murphy and Coy, 1988). After 3 h at 37°C in an air/carbon dioxide atmosphere (95-5%), the medium was removed and stored at - 2 0 ° C until assayed for hormone content.

2.6. GH RIA (radioimmunoassay) GH in media was measured by a standard double antibody RIA using components generously supplied by the NIDDK and the National Hormone and Pituitary Program, University of Maryland School of Medicine.

2. Z In vitro potency calculations Multiple deses of analog were screened in each assay. A potency was calculated by 4-point assay (Pugsley, 1946) against a GRF-(1-29)NH 2 standard also tested in multiple doses in each cell culture assay. Potencies presented in the tables are the means of the individual assay potencies + S.E.M. for each analog. The number of individual potency determinations (different cell cultures) is given in parentheses. The IC50s for antagonist analogs were calculated by weighted, non-linear regression analysis.

TABLE 1 Comparison of Chou-Fasman a-helix and turn probability values calculated for GRH-(1-29)NH 2 and [AlaS'~aS]GRH-(1-29)NH 2Residue

1 Tyr 2 Ala 3 Asp 4 Aia 5 lie 6 Phe 7 Thr 8 Asn (Ala) 9 Ser (Ala) 10 Tyr I I Arg 12 Lys 13 Val 14 Leu 15 Gly (Ala) 16 Gin 17 Leu 18 Ser 19 Ala 20 Arg 21 Lys 22 Leu 23 Lea 24 Gin 25 Asp 26 lie

GRH-(1-29)NH 2

[AlaS.,~.IS]GRH. (I-29)NH 2

Pa

P turn

Pa

P turn

i .084 !.147 1.092 !.040 0.942 0.905 0.817 0.849 0.932 0.955 1.100 1.000 1.010 1.107 0.987 1.170 1.092 1.075 1.195 1.197 1.274 !.227 i.145 !.187 1.074 1.055

0.979 0.812 0.797 0.672 0.897 1.137 1.272 1.270 1.132 0.900 0.762 0.915 0.907 0.930 1.140 0.915 0.907 1.012 0.802 0.785 0.792 0.905 0.875 0.877 0.990 0.862

1.084 1.147 1.092 1.040 1.040 1.120 1.032 1.064 1.049 0.955 1.100 1.182 1.192 1.290 1.169 1.170 1.092 1.075 1.195 1.197 1.274 ! .227 1.145 1.187 1.074 1.055

0.979 0.812 0.797 0.672 0.672 0.720 0.854 0.852 0.940 0.900 0.762 0.690 0.682 0.705 0.915 0.915 0.907 !.012 0.803 0.785 0.792 0.905 0.875 0.877 0.990 0.862

3. Results

The results of the Chou-Fasman analysis of GRH(1-29)NH 2, which are presented in table 1, clearly revealed two regions of the molecule centered around position 15 and 6-9 which have a < 1 probability of a-helix formation accompanied by a significant increase in folding probability. Substitution of Ala for Gly in position 15 (analogue II, table 2) theoretically increases a-helix probability from 0.987 to 1.169 and this modification was accompanied by a 5-fold increase in GH releasing potency. Substitution of Ala in either position 8 or 9 also greatly increased the probability of a-helix formation at the expense of /3-turn formation around position 9 (data not shown). [AIaS]GRH-(1 29)NH 2 (III) was 4 times more potet~t than the parent peptide (table 2) and the combination analogue, [AIaS.15]GRH-(1-29)NH2 (IV), was 15 times more potent. The potency of this analogue was further boosted by the incorporation of D-AIa 2 to give [D-AIa2,Ala sAS] GRH-(1-29)NH 2 (V) which was 27 times more potent. This should be compared to [D-AIa 2]GRH-(1-29)NH 2 (I) itself which was 3 times more potent. The presence of Ala in both positions 8 and 9 further increases the likelihood of helix formation from position 5 to posi-

~S2 L~\BLE 2 St~l~clures and in vitn~ potencies of G R I t ( I - 2 q } N t t , analogs on gro~th hormone secretion from 4-day cultures of rat pituitary cells.

Potencies ~means ± S.E.} ~ere calculated from individual cell culture experiments b5 4-point assay (Pugsley. !t}46). The value in parenthe~ s is lhe number of independent experiment~ using multiple doses of analog used in calcuk~ting the mean potency. A G R H standard cur~'e (potem.3' = I} ~as contained in each assay.

1 ll Iil IV V V1 VIi ViII IX X XI X|I Xlll XIV XV XVI XVll XVI|! XIX

Analog

Potency

GRH D-AIa z Ala Ds Ala s Ala s'gs D-AktZ.Aias'ts D-AlaZ,Ala s''~'t5 AlaStS.Giu'-S.Leu '~'''-; D-AiaZ.AlaSAS.GluZS,Leu zt''-7 AlaS~L. G l u . -~ . L e u . ."~ .... "" D-Ala .Leu .Ala" . G l u - . L e u - Ala :s'"" c" Aia s''~a"'~s D-Asn s D-AlaS.Ala i~ AibS,Aia ts N-MeAlaS,Ala t~ D-N-MeAlaS.Ala ts D h :'; , ~ ! . 1 5 r,,e . ~ a D-PheS.Ala Is

! 3 " 5 _+ I (4) 4 + I (13) 15 +_ 2 (15) 27 _+ I0 (8) 4q + 14 (8) 7 _+ 2 (5) 7 +_ 1 (4) 33 + 9 (6) 11.3 ___ 11.2 (5) 2 +_ i (2) 1 + 1 (2) 3 _+ |1.~ (3) 6 +_ 2 (q) 2 +_ 11.6 (3) 0.i +_ 0.04 (5) ll.{}l+ 0.{1115 (5) 3 + 1 (6) 1 +_ 0.4 (6)

From Murphy and Coy (1988).

500

400 t-.

s00

200

v

tion 10 (table 1) and [D-Ala-',AlaS"~'IS]GRH-(1-29)NH2 (Vl) was found to be 49 times more potent than GRH itself (table 2). Aib in position 8 (XV, table 2) was less effective than Ala in increasing potency (compare to analogue IV in table 2). Several analogues were made with Glu in position 25 and Leu in positions 26 and 27 which, according to Chou-Fasman analysis (not shown), should marginally increase a-helix probability in the C-terminal region. This strategy resulted in no increase in biological potency of key analogues tested (compare analogues VII, VIll, and IX in table 2). Analogues with Ala in position 7 or in position 10 to give [AIa7'S'9"tS]GRH-(129)NH 2 (XI) and [AlaS:~'m'tS]GRH-(1-29)NH2 (XII) were 2 times more potent and equipotent to GRH, respectively, representing a large loss of potency compared to analogue VI. The presence of Leu in place of Thr in position 7 was even more injurious to potency (compare analogues X and VIII in table 2). D--Amino acids in position 8 also improved potency. [D-AsnS]GRH-(I-29)NH2 (XIII, table 2) and [DAlaS,Ala~5]GRH-(1-29)NH2 (XIV)were 3 times and 6 times more potent, respectively. [D-PheS,AlatS]GRH (1-29NH 2 (XIX, table 2) was only equipotent to GRH and was less potent than its L-Phe counterpart (XVIII). Analogues incorporating either N-Me-Ala or D-N-MeAla in position 8 (analogues XVI and XVII, table 2) exhibited very low levels of GH releasing activity. Typical dose response curves for GRH, [D-AlaZ,Ala8,t5] GRH-(1-29)NH 2, and the most potent [D-Ala 2, AlaS'~'IS]GRH-(1-29)NH2 analogue are shown in fig. 2. The dose-response effects of the combined Ala-8, 9 and 15 substitutions on the inhibitory potency of a standard GRH competitive receptor antagonist, [DArg2,Leu27]GRH-(I-29)NH2, are shown in fig. 3. [DPhe2,Leu27]GRH-(1-29)NH2, a weak VIP receptor antagonist, was also included in this assay as a control analogue and, as expected, had no discernable effect on GRH-stimulated GH release. [D-ArgZ,Leu27]GRH-(1-29)NH 2 itself had an IC50 value of 2.2(+0.9) X 10 - 7 M against pituitary cell GH release stimulated by a 10 -9 M concentration of GRH. With [D-Arg 2, AlaS'tS]GRH-(1-29)NH2 this was decreased to 5.9(+2.2) x 10 -8 M and with [D-Arg2,Alaa.9.15]GRH (1-29)NH 2 to 1.7(+0.7)x 10 -8 M.

i(3o -13

-12

-I I

-10

-9

4. Discussion

Dose [Log Molarl Fig. 2. Dose-response curves of analog V. VI and G R H on GH secretion from 4-day primary cultures of rat pituitary cells. Media GH is expressed as the percentage of GH released into media in the presence of 0.1 nM somatostatin and no analog or G R H present [control]. Values are the means + S.E. of the mean responses determined for each dose (in quadruplicate) from independent assays. The data were pooled from eight independent cell culture assays.

It has been recognized for a number of years that the amphiphilic a a-helical structure of GRH peptides could theoretically be improved by the use of suitable Chou-Fasman 'enhanced a-helix forming' amino acids at suitable positions along the chain. Both Velicebeli et al. (1986) and Tou et al. (1986) made multiple substitu-

183

400

t','J

re~

[]

D-Phe-2. Leu-27- GRH

[]

D-Atg-2. Leu-27 - GRH

l~

D-Atg-2, Ala-8,15 - GRH

I~l D-Atg-2, Ala-8,9,15 - GRH

300

"e L~

200

I

O0

. Control

.

.

. -I0

.

.

.

. -9

.

.

.

.

-8

"

-7

-6

Dose ILog Mol~l Fig. 3. Antagonism of 1 nM GRH-stimulated GH release from 4-day primary cultures of rat pituitary cells by GRH analogs. Media GH is expressed as the percentage of GH released into media in the presence of 0.1 nM somatostatin and no analog or GRH present [basal]. Control is GH release in the presence of 0.1 nM SRIF and 1.0 nM GRH expressed as a percentage of basal secretion. Values are the means_+S.E. of the mean responses determined for each dose (in quadruplicate) from independent assays.The data were pooled from four independen! cell culture assays.

tions with this in mind which, as in the present paper, included Ala replacements in positions 15 and 9. However, although potency was maintained, little actual enhancement was realized presumably because the multiple replacements were not uniformly beneficial to biological activity. Single amino acid replacements using this strategy have had more success, most notably the use of various amino acids substituted in place of Gly in position 15 (Felix et al., 1988). Ala appears to be by far the best a-helix-inducing residue in a number of recent studies (Lyu et al., 1990; O'Neil and DeGrado, 1990) and was also the best choice in position 15 of G R F (Felix et al., 1988). In the present study, it produced the 5-fold increase in potency exhibited by [AlanS]GRH-(1-29)NH2 in table 2 which agrees well with previous in vitro potency estimates (Felix et al., 1988). Theoretically, as can be seen from table l, the 6-10 region of G R H is the most conformationally ambiguous with a /3-turn centered around Asn s being predicted by the C h o u - F a s m a n calculations. This is supported by the increased potency of [D-AsnS]GRH-(1 29)NH 2 both in vivo (Coy et ai., 1985) and in vitro (table 2) which would seem to be caused by increased receptor affinity rather than proteolytic resistance. DAmino acids are known to stabilize fl-turns and in-

crease potency. Examples of this include iuteinizing hormone-releasing hormone (LH-RH)'superagonists', such as [D-Trp~']LH-RH (Coy et al., 1976), and also [D-Trp~]somatostatin (Rivier et al., 1975), and [DPhea]glucagon (Sueiras-Diaz, 1984). However, in the present study, D-Phe s and analogues containing either steroeisomer of N-Me-AIa (another turn-favoring amino acid) in position 8 were substantially less potent (table 2) which argues against the turn hypothesis. Indeed, the substantial increase in C h o u - F a s m a n helix probabilities which are closely paralleled by substantial potency increases in Ala 8 and Ala~ analogues now seem to demonstrate quite conclusively that it is the a-helical characteristics of the molecule which are important for high receptor affinity and furthermore that, in view of the data in table 1, that these could very well extend from position 2 through to the C-terminus of the chain. It has also been shown previously that certain potency-enhancing modifications to G R H could be combined with additive effects. Most notable of these have been combinations of D-AIa 2 and position 15 enhancements (Felix et al., 1987). In vitro, D-AIa in position 2 results in a 3-fold increase in potency (table 2) and, when combined with Ala uS, this is reported to increase to around 6-fold (Felix et al., 1987). Likewise in table 2,

t,~sition ..," S, 0 and t'~, substitutions were all apparently additive in terms of their biological effects and the most potent a n a l o g u e , [D-Alae,AlaS"~'IS]GRH-(I 2'4}NH, (VI, table 2), a p p e a r s to be the most effective G R H agonist analogue to have been r e p o r t e d and tested in an in vitro assay system thus far. Extension of this Ala-substitution strategy to either positions 7 or 10 {analogues XI and XII, table 2) resuRed in substantial losses of potency despite the calculated high retention of C h o u - F a s m a n helix forming ability (not shown). This is, however, to be expected since Thr ~ can be considered to be one of the most essential amino acids and is common to this position in all members of the G R H - s e c r e t i n - g | u c a g o n - V I P group of peptides. Position 1{} is in an hydrophilic region of the amphiphilic helix (see fig. 1) and could not be expected to accept the increased hydrophobicity associated with Ala substitution for Tyr. A b r a n c h e d side-chain k e u residue in position 7 of [D-Ala2,keu 7, A l a S ~ S ] G R H - ( I - 2 9 ) N H , ( X ) w a s also not well tolerated and resulted in about a 2{|-fold loss of potency (compare to analogue VIII, table 2). Wc were also interested in seeing w h e t h e r the same substitution strategy would increase the potency of a G R H competitive receptor antagonist. The best of the few which had thus far been r e p o r t e d a p p e a r e d to be [Ac.Tyr ~,D.Arg e ]G RH-( 1-29)N H , ( R o b b e r e c h t et al., 1985) which inhibited G R H - s t i m u l a t e d adenylate cyd a s e activity in rat pituitary cells with a low potency in the p.M range. This analogue was also reported (Lumpkin et al., 19"89; Lumpkin and M c D o n a l d , 1989) to readily block e n d o g e n o u s G R H activity after injection in the rat. In the present study, [D-Arg2,Leu 27] G R H - ( I - 2 9 ) N H , was a similarly effective antagonist on G R F - s t i m u l a t e d G H release from rat pituitary cells (fig. 2) and was used for further modification. Substitution of Ala s and Ala ~s to give [D-Arg-',AlaS'IS]GRH ( I - 2 9 ) N H , resulted in almost a 5-fold decrease in its ICs, value and further substitution of Ala '~ in the latter peptide gave a f u r t h e r 3-fold decrease in potency down to 17 nM. This is a far higher level of antagonist potency than recently achieved ( H o c a r t et al., 1990) using the reduced peptide bond r e p l a c e m e n t of the normal peptide bond between the 9 and 10 positions of G R H - ( 1 - 2 9 ) N H ~. The effects of helix e n h a n c e m e n t on biological activity were qualitatively similar in both the agonist and antagonist series and this suggests that the agonism enhancing effects are due to increased receptor affinity rather than an increased efficacy of r e c e p t o r activation. Recently reported (Sato et al., 1990) N-terminal alterations to [ D - A r g 2 ] G R H - ( 1 - 2 9 ) N H 2 which also result in enhanced antagonist potencies suggest that further combinations of these and the modifications described in the present p a p e r could result in additional worthwhile potency improvements.

Acknowledgements We would like to thank V. Mackey and E. Yauger for their expert technical assistance. This research was supported by NIH Grant DK-31|167.

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