CA1341070C - Pulmonary surfactant protein and related polypeptide - Google Patents

Abstract

The present invention relates to a human SP18 monomer protein-related polypeptide useful in forming a synthetic pulmonary surfactant. The present invention also relates to a method of treating neonatal respiratory distress syndrome comprising administering a therapeutically effective amount of synthetic pulmonary of the present invention. Further contemplated by the present invention is a composition containing human SP18 monomer and human SP18 dimer but no other pulmonary surfactant proteins. A recombinant DNA molecule capable of expressing, without post-translational proteolytic processing, mature human SP18 monomer, and methods of using the recombinant DNA
molecule are also contemplated.

Description

PULMCINARY SURFACTANT PROTEIN
ANI) RELATED POLYPEPTIDE
Technical Field The present invention relates to SP18 monomer-related polypeptides useful in forming synthetic pulmonary surfactants.
The present invention also relates to a recombinant nucleic acid molecule carrying ;e stuuctural gene that encodes human SP18 monomer protein and the use of such a recombinant molecule to produce human SP18 monomer.
Background Pulmonary surfactant (PS} lines the alveolar epithelium of mature mammalian lungs. Natural PS has been described as a "lipoprotein complex" because it contains both phospholipids and apoproteins that interact to reduce surface tension at the lung air-liquid interface.
Since the discovery of pulmonary surfactant, and the subsequent finding that. its deficiency was the primary cause of neonatal respiratory distress syndrome (RDS}, a number of studies have been directed towards developing effective surfactant replacement therap~,r for affected individuals, particularly infants, using exoc~enou.s PS. For instance, improvements in lung function as measured by a decrease in mean airway pressure and oxygen requirements have been demonstrated using exogenous surfactants :in human pre-term infants. See Hallman, et al, Pediai=rics, 71:473-482 (1983); Merritt, et al, J. Pediatr., 108:741-745 (1986); Hallman, et al, _J.
Pediatr., 10E;:963-969 (1985); Morley, et al, Lancet, i:64-68 (198:L); Merritt, et al, New England J. Med., 315:785-790 (1986), Smyth, et al, Pediatrics, 71:913-917 (1983); F;nhorning, et al, Pediatrics, 76:145-153 (1985); Fujiwara, et al, The Lancet, 1:55-59 (1980);
Kwong, et al, Pediatrics, 76:585-592 (1985); Shapiro, et al, Pediatrics, 76:593-599 (1985); Fujiwara, in "Pulmonary Surfactant", Robertson, B., Van Golde, L.M.G., Batenburg J. (eds), Elsevier Science Publishers, P.,msterdam, pp. 479-503, (1984).
From a pharmacologic point of view, the optimal exogenous PS to use in the treatment of RDS would be one completely synthesized in the laboratory, under controlled and sterile conditions, with negligible batch-to-batch variability in properties. To minimize the possibility of immunologic complications, the apoprotein component of an exogenous PS should be identical to that :Found in humans. Unfortunately, the composition of naturally occurring PS is complex, and the art has not yei: identified all of the biochemical components that generate the biophysical properties needed for high physiologic activity in lungs. In particular, the ari: has failed to characterize all of the apoproteins present in natural PS or identify the function of the PS apoproteins presently known.
It should be noted that the literature on PS
apoproteins and their roles in surfactant function is complex, inconsistent and sometimes contradictory because heterogenous apoprotein preparations were used in many studies. To date, the art has not definitively established the number of different apoproteins present in natural PS.
Of particular interest to the present invention is the use of a low molecular weight (LMW) human PS-associated aF>oprotein as a component in an exogenous surfactant. Several studies have attempted to isolated or define human PS LMW apoproteins using biochemical techniques. See, for example, Phizackerley, et al, Biochem. J., 183:731-736 (1979), Revak, et al, Am. :Rev. Resp. Dis., 134:1258-1265 (1986), Suzuk:i, et al, Eur. J. Respir. Dis., 69:335-345 (1986), Taeusc;h, et al, Pediatrics, 77:572-581 (1986), Yu, et al, Biochem. J., 236:85-89 (1986), Whitsett, et al, Pediatric Res., 20:460-467 (1986), Whitsett, et al, Pediatric Res., 20:744-749 (1986), Takahashi, et al, Biochem. Bio hys. Res. Comm., 135:527-532 (1986)" Suzuki, et al, Exp. Lung. Res., 11:61-73 (1986), Curstedt, et al, Eur. J. Biochem., 168:255-262 (1987),, Notter, et al, Chem. Phys. Lipids, 44:1-17 (1987), and Phelps, et al, Am. Rev. Respir.
Dis., 135:1112-111'7 (1987).
Recently, the art has begun to apply the methods of recombinant DNA technology to overcome the problems associated with not: being able to isolate to homogeneity the individual LMW PS apoproteins. For instance, Glasser, et al, Proc. Natl. Acad. Sci., U.S.A., 84:4007-4011 (1987) reported a cDNA derived sequence of amino acid residues that forms at least a portion of a :human precursor protein from which at least one mature LbZW apoprotein, which they designated SPL (Phe), is formed. While Glasser, et al were not able to determine t:he carboxy-terminal residue of SPL(Phe), and therefore were not able to identify its complete sequence, they did predict that mature SPL(Phe) was ,bout 60 amino acids in length.

Jacobs, et al, J. Biol. Chem., 262:9808-9811 (1987) have described a cDNA and derived amino acid residue sequE:nce for a human precursor protein similar to that described by Glasser, et al, su ra. However, according to Jacobs et al. the mature LMW apoprotein, which they designated PSP-B, formed from the precursor would be 76 amino acid residues in length. In addition, Jac:obs, et al, noted that it was not clear that any PS a.poprotein derived from the reported precursor protein 'was present in the surfactant preparations that :had been studied clinically by others.
From the foregoing it can be seen that the literature contains multiple nomenclature for what is apparently the same PS apoprotein. Therefore, for ease of discussion" the mature apoprotein derived from the precursor protein described by Glasser, et al, supra, and Jacobs, et al, supra, will be referred to herein generically as "SP18", with the monomeric and dimeric forms being referred to as "SP18 monomer" and "SP18 dimer", respe:ctively, when appropriate.
The canine SP1.8 precursor has been described by Hawgood, et al, Proc. Natl. Acad. Sci. U.S.A., 84:66-70 (1987) and Schi7_ling, et al, International Patent Application W~D 86/03408. However, it should be noted that both those studies suffered the same inability to define the mature, biologically active form of SP18 as the Glasser, ~~t al, su ra, and Jacobs, et al, su ra, studies.
Warr, et al, Proc. Natl. Acad. Sci., U.S.A., 84:7915-7919 (1987) describe a cDNA derived sequence of 197 amino acid residues that forms a precursor protein from which a mature LMW apoprotein, they designate as SPS, i.s formed. Like the studies attempting to describe SP18, Warr, et al, were unable to determine the c:arboxvy terminal residue of the mature protein formed from the precursor protein sequence, and thus were not able to definitively characterize SP5.
Because the amino acid residue sequence of the precursor protein reported by Warr, et al, is different from that reported by Glasser, et al, and ;Jacobs, et al, it therefore appears that the art has determined l~hat natural PS contains at least two LMW
apoproteins. However, l~he biologically active forms of those proteins has remained undetermined.
Summary It has row been found that human SP18 is a homodimeric protein (SP18 dime~r) whose mature subunit protein (SP18 monomer) displays an apparent mo7Lecular weight of about 9,000 daltons when determined by sodium do<~ecyl sulfate polyacrylamide gel electrophoresis {SDS-PAGE).
It has also been discovered that human SP18 can function as an active ingredient in a synthetic pulmonary surfactant in the absence of other previously identified pulmonary surfactant proteins.
In addition, t:he carboxy-terminal amino acid residue sequence of the mature ruuman SP18 monomer protein has been determined, and thus it:> naturally occurring form has now been discovered.
Thus, the pre~cent invention contemplates a composition comprising substantially isolated or substantially pure human SP18 monomer.
B

~ 341 07 0 5a The preaent invention therefore provides a polypeptide consisting essent:lally of at least 10 amino acid residues and no more than about 60 amino acid residues, said polypeptide including a sequence having alternating hydrophobic and hydrophilic amino acid residue regions represented by the formula (ZaUb)cZd' wherein:
Z is a hydrophilic amino acid residue independently selected from the group consisting of R, D, E, and K;
U is a hydrol>hobic amino acid residue independently selected from the group consisting of V, T, L, C, Y and F;
a has an average v<~lue of about 1 to about 5;
b has an average v<alue of about 3 to about 20;
c is 1 to 10; and d is 0 to 3;
said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, for~riing a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.
The invention also provides a polypeptide having an amino acid residue sequence selected from the group consisting of:
KLL~LLKLLLLKLLLLKLLLLK , KLLLLLLLLKLLLLLLLLKLL, KKLLLLLLLKKLLLLLLLKKL.
DLLLLDLLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLI~RLLLLLLLLRLL, RRLLLLLLhRRLLLLLLLRRL, RLLLLCLLI~RLLLLCLLLR , .'~ .~.

1 34 ~ 07 0 5b RLhLLCLLLRLLLLCLLLRLL, and RLhLLCLLLRLLLLCLLLRLLLLCLLLR.
The inv~~ntian additionally provides a composite polypeptide of at least 10 and no more than 60 amino acid residues consisting essentially of an amino terminal sequence and a carboxy terminal sequence wherein:
said amino te~rmina:l sequence has at least 10 amino acid residues and has a composite hydrophobicity of less than 0; and said carboxy terminal sequence is characterized as consisting essentially of an amino acid residue sequence represented by the formula:
-RLVLRCSMDDz, wherein Z is an integer having a value of 0 or 1, such that when Z
is 0 the D residue: to which it is a subscript is absent and when Z
is 1 the D residue: to which it is a subscript is present;
said composite polypeptide, when admixed with a pharmaceutically accept<~ble phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of i:he phospholipid alone.
The invention further provides synthetic pulmonary surfactants comprising one or more pharmaceutically acceptable phospholipids admixed with such polypeptides.
The invention also provides commercial packages comprising such polypept:ides or synthetic pulmonary surfactants present in therapeutically effective amounts together with instructions for use to treat respiratory distress syndrome. The use of such compounds and compositions to treat respiratory distress syggndrome is a further aspect of the invention.

~ 341 07 0 SC
In preferred embodiments of the invention described above:
(a) Z is independently selected from the group consisting of R and K;
(b) U is in<iepend~ently selected from the group consisting of V, I, L, C and F, especially L and C;
(C) d is 1;
(d) C is 4;
(e) a is 1 or 2;
(f) b has an average value of about 3 to about 9; or (g) in the ecequenc:e of the formula (ZaUb)oZd' Z i.s indEapendently selected from R and K;
U i.s inde:pendently selected from L and C;
a is 1 or 2;
b h,as an average value of about 3 to about 8;
c is 1 to 10; and d is 0 to 2.
Also contemplated by the present invention is a DNA
segment consisting essentially of a DNA sequence encoding human SP18 monomer protein.
Another embodiment of the present invention is a recombinant nucleic acid molecule comprising a vector B

operatively linked to a structural gene capable of expressing, without post-translational proteolytic modification, human SP18 monomer protein.
Also contemplated is a method of preparing human SP18 monomer protein comprising:
(a) ini.tiati.ng a culture, in a nutrient medium, of mammalian cells transformed with a recombinant DNA
molecule comprising a vector operatively linked to a structural gene ca~.pable of expressing, without post-translationa:L proteolytic modification, human SP18 monomer;
(b) maintaining said culture for a time period sufficient for said cells to express human SP18 monomer protein from said recombinant DNA molecule;
and (c) recovering said protein from said culture.
Further contemplated is a polypeptide consisting essentially of at least 10 amino acid residues and no more than about 60 amino acid residues corresponding in sequence t:o the amino acid residue sequence of human SP18 monomer, said polypeptide, when admixed with a pharma:ceuti~cally acceptable phospholipid form a synthetic surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.
In another embodiment, the subject invention contemplates a syn~~hetic pulmonary surfactant comprising a pharmaceutically acceptable phospholipid admixed with a pol~~peptide consisting essentially of at least 10 amino acid residues and no more than about 60 amino acid residues corresponding in sequence to the amino acid residue sequence of human SP18 monomer.
A further embc>diment of the subject invention is a method of treating respiratory distress syndrome comprising administering a therapeutically effective ?34? 070 amount of a synthea is pulmonary surfactant, said surfactant c~~mpri:~ing a pharmaceutically acceptable phospholipid admix;ed with an effective amount of a polypeptide ~~onsi~;ting essentially of at least 10 amino acid rE~sidue:s and no more than about 60 amino acid residues corresponding in sequence to the amino acid residue sequence of human SP18 monomer, said polypeptide, when admixed with a pharmaceutically acceptable phospholipid forms a synthetic surfactant having a surf=actant activity greater than the surfactant aca ivaty of the phospholipid alone.
Another embodiment of the subject invention is a method of treating respiratory distress syndrome comprising administering a therapeutically effective amount of a ~;ynthetic pulmonary surfactant, said surfactant comprising an effective amount of either substantially isolated human SP18 monomer or substantially pure human SP18 monomer admixed with a pharmaceutically acceptable phospholipid.
Brief Descri tion of Drawin s Figure 1 illustrates a 750 nucleotide cDNA
sequence (top line,) and deduced amino acid residue sequence (bottom lanes). The number to the right of each line of nucleotides represents the numerical position in the sequence of the nucleotide at the end of each line. The nucleotides are grouped into codons, 15 codons per line, with the amino acid residue coded for by each codon shown in triple letter code directly below the codon. The numerical position of some residues in the amino acid residue sequence encoded by the cDNA is shown below the residues. T_he amino-terminal amino acid residue of mature human SP18 monomer is Ph~~ (enc:oded by nucleotides 187-189) and is designated re;sidue~> number 1. The carboxy-terminal 1 34~ 07 0 _8_ amino acid residuE=_ is Asp at residue position 81 (encoded by nucleotides 427-4a?9). ;~1 structural gene encoding mature SP18 monomer therefore conta:i:ns 81 codons and has a nucleotide sequence that corz-esponds to nucleotides 187-429.
Figure 2. illustrates the protein elution profile of PS apoproteins from a B:io-Sil HA (silicic acid) column.
Results of Pierce BCA protein assay (solid line) and phospholipid anal~.rses (broken line) are shown for selected fractions. Two mi.llilit~ers (ml) were collected per fraction.
Positive protein assay in fractions 28 to 33 is due to the presence of phospholipids.
Figure 3 illu:~trates a Silver-stained SDS-PAGE of low molecular weight (LMW) F?S apoproteins. Lanes A and D show a sample after silicic acid or Sephadex* LH-20 chromatography;
both LMW proteins are px-esent. Lanes B, C, E and F show the resolution of SP18 (lanes B and E) and SP9 (lanes C and F) following chromatography on Sephadex* LH-60. Molecular weight standards are shown in lane G. Lanes A-C are unreduced samples, lanes D-F contain identical samples reduced with (3-mercaptoethanol prior to electrophoresis.
Figure 4 illu~>trates inflation and deflation pressure/volume curves c>f fetal rabbit lungs 30 min after intratracheal instillation of 100 ul of saline (open circles), 2 mg phospholipids (PL) DPPC:PG, 3:1 (closed circles), PL + 10 ug SP9 (open squares) PL + 10 ug SP18 (closed squares), or 2 mg natural human surfactant (closed triangles). Data are expressed as the mean of 4 animals ~ one standard deviation.
Figure 5 illustrates fetal rabbit lung tissue samples (x125 magnification, hernatoxyling-eosin stain) following treatment with saline (A.), natural human ~~;;~ *Trade-mark surfactant (:B) , phospholipids DPPC:PG (C) or phospholipid;s plu~~ LMW apoproteins (SP9 + SP18) (D) .
Figure Ei illustrates the surfactant activity of exemplary po:Lypept.ide containing synthetic surfactants of the present invention. Surfactant activity was determined b~T meas.uring the pressure gradient across an air/liqui<i interface using the pulsating bubble technique. ~~he pressure gradient (0P) across the surface of tree bubble is the absolute value of the pressure recorded in centimeters of HzO. The results obtained for each synthetic pulmonary surfactant are identified by the polypeptide in the surfactant.
Results obtain for surfactants consisting of phospholipid alone (i.e., with no peptide or protein admixed therewith) are identified as PL. The results obtained using a control peptide having only 8 amino acid residues. and having a sequence corresponding to human SP18 monomer residues 74-81 (p74-81) are also shown. The data time points shown were obtained at 15 seconds, 1 minute .and 5 minutes.
Figure 7 is a series of two graphs that illustrate the results of a svtatic compliance study of exemplary synthetic surfactants of this invention using the fetal rabbit model previously described in Revak, et al, Am. Rev. Respi:r. Dis., 134:1258-1265 (1986).
Following instillai~ion of a synthetic surfactant or control into the trachea, the rabbit was ventillated for 30 minutes prior to making static compliance measurements. The "x" axis represents the pressure in cm of water, while the "y" axis represents the volume in ml/kg of body weight. The graph on the left represents values <it inflation and the graph on the right represents de=flation 'values. The results for the following tested surfactants are illustrated:
natural surfactant (open square with a dot in the center), phospholipid (PL) with 7$ p52-81 (a polypeptide corresponding to residues 52 to 81 of SP18) (closed diannonds); PL with 3$ P52-81 (closed squares with white dot in center); PL with 7$ p36-81 5 (open diamonds); PL with 3$ p66-81 (closed squares);
PL with 3$ pl-15 I;open squares) and PL control (closed triangles).
Figure 8 is a series of two graphs that illustrate the results «f a ~;tatic compliance study of exemplary 10 synthetic su:rfactamts of the invention. The procedure was performed as dlescribed in Figure 7 except that a different in;;tilla.tion procedure was used. The "x"
and "y" axis and right and left graphs are as described in Figure 7. The results for the following tested surfacaants are illustrated: natural surfactant (open squares with a dot in the center);
phospholipid (PL) with 10$ p51-81 (closed diamonds);
PL with 10$ p51-76 (closed squares); and PL (closed triangles).
Detailed Description of the Invention A. Definitions Amino Acid: All amino acid residues identified herein are in the natural L-configuration.
In keeping with standard polypeptide nomenclature, _J.
Biol. Chem., 243:3557-59, (1969), abbreviations for amino acid re~sidue~s are as shown in the following Table of Correspondence:

~ 341 07 0 TABLE OF CORRESPONDENCE
SYMBOL AMINO ACID
-T~Pt't'PW ~-T ~i-~~"..
Y Tyr L-tyrosine G Gly glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P Pro L-proline K Lys L-lysine H His L-histidine Q Gln L-glutamine E Glu L-glutamic acid Trp L-tryptophan R Arg L-arginine D Asp L-aspartic acid N Asn L-asparagine C Cys L-cysteine It should be noted that all amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino--terminus to carboxy-terminus.
Furthermore, it should be noted that a dash at the beginning or end of: an amino acid residue sequence indicates a bond to a radical such as H and OH
(hydrogen and hydroxyl) at the amino- and carboxy-termini, respectively, or a further sequence of one or more amino acid residues. In addition, it should be noted that a virgu7.e (/) at the right-hand end of a 12 ~ 34 1 07 0 .
residue sequEsnce indicates that the sequence is continued on the next line.
Polype~t:ide and Peptide: Polypeptide and peptide are terms usE~d interchangeably herein to designate a linear serie:~ of no more than about 60 amino acid residues connected one to the other by peptide bonds between the ~ilpha-amino and carboxy groups of adjacent residues.
Protein: Protein is a term used herein to designate a 1_inear series of greater than about 60 amino acid residues connected one to the other as in a polypeptide.
Nucleotide: a monomeric unit of DNA or RNA
consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphaite group bonded to the 3' or 5' position of the pentose it is referred to as a nucleotide.
Base Pair (bp): A partnership of adenine (A) with thymine (T) , or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
B. SPlf3 Monomer-Containing Com ositions The present invention contemplates a SP18 monomer-containing composition (subject protein composition) wherein the SP18 monomer is present in either substa.ntial7_y isolated or substantially pure form. By "is~~latecl" is meant that SP18 monomer and SP18 dimer are pre~~ent as part of a composition free of other alveolar surfactant proteins.
By "'substantially pure" is meant that SP18 monomer is present as part of a composition free of ~34~070 other alveolar surfactant proteins and wherein less than 20 percE:nt, preferably less than 10 percent and more preferably less than 5 percent, of the SP18 monomer presE~nt is in homodimeric form, i.e., present as part of SF~18 dimer.
Preferably, a SP18 monomer-containing composition of the present invention contains human SP18 monomer. More preferably, a SP18 monomer-containing cc>mposition contains SP18 monomer having an amino acid residue sequence corresponding to the amino acid residue sequence shown in Figure 1 from about residue position 1 to at least about residue position 75, preferably to .at least about position 81. More preferably, a. SP18 monomer used to form a subject protein composition corresponds in sequence to the sequence shown in :Figure 1 from residue position 1 to residue position 81.
Pre:Eerabl.y, the amino acid residue sequence of a SP18 monomer :in a subject SP18 monomer-containing composition corresponds to the sequence of a native SP18 monomer. However, it should be understood that a SP18 monomer used i~o form a protein composition of the present invention need not be identical to the amino acid residue sequence of a native SP18 monomer, but may be subject to ~;rarious changes, such as those described hereinbe:Low for a polypeptide of this invention, so long as such modifications do not destroy surfactant activity. Such modified protein can be produced, as is well known in the art, through, for example, genom:ic site-directed mutagenesis.
"Surfactant activity" for a protein or polypeptide is defined as the ability, when combined with lipids, either alone or in combination with other proteins, to exhibit activity in the in vivo assay of Robertson, Zuni, 1!58:57-68 (1980). In this assay, the ? 341 070 sample to be assessed is administered through an endotracheal tube to fetal rabbits or lambs delivered prematurely by Caesarian section. (These "preemies"
lack their own PS, and are supported on a ventilator.) Measurements of lung compliance, blood gases and ventilator pressure provide indices of activity.
Preliminary assessment of activity may also be made by an in vitro assay, for example that of King, et al, Am. J. Physiol., 223:715-726 (1972), or that illustrated below which utilizes a measurement of surface tension at a air-water interface when a protein or polypeptide is admixed with a phospholipid.
C. Nucleic Acid Segments In living organisms, the amino acid residue sequence of a protein or polypeptide is directly related via t:he genetic code to the deoxyribonucleic acid (DNA) sequence of the structural gene that codes for the protein. Thus, a structural gene can be defined in teams of the amino acid residue sequence, i.e., protein or polypeptide, for which it codes.
An important and well known feature of the genetic code is its redundancy. That is, for most of the amino acids used to make proteins, more than one coding nucleotide triplet (codon) can code for or designate a particular amino acid residue. Therefore, a number of cliffer~ent nucleotide sequences may code for a particular amino acid residue sequence. Such nucleotide se:quenc~es are considered functionally equivalent since they can result in the production of the same amino acid residue sequence in all organisms.
Occasionally, a methylated variant of a purine or pyrimidine ma.y be incorporated into a given nucleotide sequence. However, such methylations do not affect the coding re:latio:nship in any way.

15 1 3t 1 p~ ~
A DNA segment of the present invention is characterized as consisting essentially of a DNA
sequence that. encodes a SP18 monomer, preferably human SP18 monomer.. That is, a DNA segment of the present invention forms a structural gene capable of expressing a SP18 monomer. While the codons of the DNA segment need not be collinear with the amino acid residue sequence of SP18 monomer because of the presence of an intron, it is preferred that the structural gene be capable of expressing SP18 monomer in mature form, i.e., without the need for post-translationa7. proteolytic processing. Preferably, the gene is present as an uninterrupted linear series of codons where each codon codes for an amino acid residue found in a SP18 monomer, i.e., a gene containing no introns.
Thus, a L>NA segment consisting essentially of the sequence shown in Figure 1 from about nucleotide position 187 to about nucleotide position 426, preferably to about nucleotide position 429, and capable of expressing SP18 monomer, constitutes one preferred embodiment of the present invention.
DNA segments that encode SP18 monomer can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteuccci, et al, J. Am. Ch.em. Soc., 103:3185 (1981) . Of course, by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence.
Also contemplated by the present invention are ribonucleic acid (RNA) equivalents of the above described DNA. segments .

16 ~v~~ 070 D. Recombinant Nucleic Acid Molecules The recombinant nucleic acid molecules of the present invention can be produced by operatively linking a vector to a nucleic acid segment of the present invention.
As used herein, the phase "operatively linked"
means that tree subject nucleic acid segment is attached to t:he vector so that expression of the structural gene formed by the segment is under the control of the vector.
As 'used herein, the term "vector" refers to a nucleic acid molecule capable of replication in a cell and to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment. Vectors capable of directing the expression of a structural gene coding for SP18 monomer are referred to herein as "expression vectors." Thus, a recombinant nucleic acid molecule (rDNA or rRNA.) is a hybrid molecule comprising at least two nucleotide sequences not normally found together in nature.
The choice of vector to which a nucleic acid segment of the pre:;ent invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed, these being limitations :inherent in the art of constructing recombinant nucleic acid molecules. However, a vector contemplated by thE~ present invention is at least capable of directing the replication, and preferably also expression, of SP18 monomer structural gene included in a nuclE~ic acid segment to which it is operatively linked.
In preferred embodiments, a vector contemplated by thE~ present invention includes a procaryotic i:eplicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of an rDNA molecule extrachromosomally in a procaryotic; host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, those embodiments that include a procaryotic replicon also include a gene whose expres~;ion confers drug resistance to a bacterial host transformed therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
Tho:;e veca ors that include a procaryotic replicon can also :include a procaryotic promoter capable of directing the expression (transcription and translation) of a SP18 monomer gene in a bacterial host cell, such as E. coli, transformed therewith. A
promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription i.o occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such vector plasmids are pUCB, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA) and pPL and pKK223 available from Pharmacia, Piscataway, N.J.
Expression vectors compatible with eucaryotic cells, preferably those compatible with vertebrate cells, can also be used to form an rDNA molecule of the present invention. Eucaryotic cell expression vectors are well known in the art and are available from several commei:cial sources . Typically, such vectors are providE:d containing convenient restriction sites for insertion of the desired DNA segment.
Typical of such veca ors are pSVL and pKSV-10 18 ~ 341 0~ 0 (Pharmacia), pBPV-~1/pML2d (International Biotechnolog:i.es, I:nc.), and pTDT1 (ATCC, #31255).
In preferred embodiments, a eucaryotic cell expression vector used to construct an rDNA molecule of the present invention includes a selection marker that is effects ive in a eucaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin i:esistance, i.e., the neomycin phosphotransf:erase (neo) gene. Southern, et al, _J.
Mol. Appl. Genet., 1:327-341 (1982).
The use of retroviral expression vectors to form a recomx>inant nucleic acid molecule of the present invention is also contemplated. As used herein, the term ":retroviral expression vector" refers to a nucleic acid molecule that includes a promoter sequence derived from the long terminal repeat (LTR) region of a retrov:irus genome.
In preferred embodiments, the expression vector is a retrov:iral expression vector that is replication-incompEatent in eucaryotic cells. The construction and use of retroviral vectors has been described by Sorge,, et al, Mol. Cell. Biol., 4:1730-37 (1984) .
A variety of methods have been developed to operatively link nucleic acid segments to vectors via complementary cohe:>ive termini. For instance, complementary homopolymer tracts can be added to the nucleic acid segment to be inserted and to a terminal portion of the vector nucleic acid. The vector and nucleic acid segment are then joined by hydrogen bonding between thE: complementary homopolymeric tails to form a recombinant nucleic acid molecule.
Synthetic linkers containing one or more restriction sites provide an alternative method of 19 ~34a 070 joining a nucleic acid segment to vectors. For instance, a I)NA segment of the present invention is treated with bacte:riophage T4 DNA polymerase or _E.
coli DNA polymerase I, enzymes that remove protruding, 3', single-stranded termini with their 3'-5' exonucleolytu c activities and fill in recessed 3' ends with their polymerizing activities. The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacte:riophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA
segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synt=hetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International BiotE:chnologies, Inc., New Haven, CT.
Also contemplated by the present invention are RNA equivalents of the above described recombinant DNA molecules.
E. Transformed Cells and Cultures The present invention also relates to a host cell transformed with a recombinant nucleic acid molecule of t:he prE:sent invention, preferably an rDNA
capable of expressing an SP18 monomer. The host cell can be either procaryotic or eucaryotic.
"Cel.ls" or "transformed host cells" or "host cells" are often u~>ed interchangeably as will be clear 134~~~0 from the context. These terms include the immediate subject cell, and, of course, the progeny thereof. It is understoo~3 that. not all progeny are exactly identical to the parental cell, due to chance 5 mutations or differences in environment. However, such altered progeny are included when the above terms are used.
Bacteria:L cells are preferred procaryotic host cells and typically are a strain of E. coli such 10 as, for example, the E. coli strain DH5 available from Bethesda Research Laboratories, Inc., Bethesda, MD.
Preferred euc:aryotic host cells include yeast and mammalian ce7_ls, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic 15 cell line. Preferred eucaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61. and NIH Swiss mouse embryo cells NIH/3T3 available frc>m the ATCC as CRL 1658. Transformation of appropriate cell hosts with a recombinant nucleic 20 acid molecules of t:he present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of procaryotic host cells, see, for example, Cohen, et al, Proc . Nat l . Acad. Sci . USA, 69 : 2110 ( 1972 ) ; and Maniatis, et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). With regard to transformation of vertebrate cells with recombinant nucleic acid molecules containing retroviral vectors, see, for example, Sorge, et al, Mol. Cell. Biol., 4:1730-37 (1984); Graham, et al, Virol., 52:456 (1973); and Wigler, et al, Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979) .
Successfully transformed cells, i.e., cells that contain a recombinant nucleic acid molecule of ~34~ ono the present :invent.ion, can be identified by well known techniques. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce monoclonal colonies. Cells from those coloniE:s can. be harvested, lysed and their DNA
content exam_~ned for the presence of the rDNA using a method such <is that described by Southern, J. Mol.
Biol., 98:50:3 (1975) or Berent, et al, Biotech., 3:208 (1985) .
In addition to directly assaying for the presence of x-DNA, successful transformation can be confirmed by well known immunological methods when the rDNA is capable of directing the expression of an SP18 monomer. For example, cells successfully transformed with an expression vector operatively linked to a DNA
segment of the present invention produce proteins displaying SP18 monomer antigenicity. Thus, a sample of a cell culture suspected of containing transformed cells are harvested and assayed for human SP18 using antibodies specific for that antigen, the production and use of such anitibodies being well known in the art.
Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culi~ure of those cells, preferably a monoclonal (clonal:Ly homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. ~?referably, the culture also contains a protein displaying SP18 monomer antigenicity, and rnore preferably, biologically active SP18 monomer.
Nuti:ient media useful for culturing transformed host cells are well known in the art and can be obtained from several commercial sources. In embodiments wherein the host cell is mammalian, a "serum-free" medium is preferably used.
F. Rec:ombin,ant Methods for Producin SP18 Another .aspect of the present invention pertains to a3 method for producing SP18, preferably human SP18 mc~nomer~. The method entails initiating a culture comp~rising~ a nutrient medium containing host cells, preferably human cells, transformed with a rDNA
molecule of t:he present invention that is capable of expressing SF?18 monomer. The culture is maintained for a time pE:riod sufficient for the transformed cells to express SF~18 monomer. The expressed protein is then recovered from the culture.
Methods for recovering an expressed protein from a culture are well known in the art and include fractionation of the protein-containing portion of the culture using well known biochemical techniques.
For instance, the methods of gel filtration, gel chromatography, ultrafiltration, electrophoresis, ion exchange, affinity chromatography and the like, such as are known for protein fractionations, can be used to isolate the expressed proteins found in the culture. In addition, immunochemical methods, such as immunoaffinity, inununoadsorption and the like can be performed using we:l1 known methods.
Also contemplated by the present invention is an SP18 monomer produced by a recombinant nucleic acid method described herein.
G. Pol~~peptides A polypep~tide of the present invention (subject polypeptide) is characterized by its amino acid residue sequence and novel functional properties.
A subject polypept:ide when admixed with a ~34~ 070 pharmaceutically acceptable phospholipid forms a synthetic pu:Lmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone (as indicated by a lower OP as shown in Figure 6 <ind a higher volume per given pressure as shown in Figures 7 and 8).
As seen in Figure 1, SP18 has a large hydrophobic region (residues 1 to about 75), followed by a relatively snort hydrophilic region at the carboxy terminus (re~~idues 76 to 80, or 81). In referring to amino acid residue numbers of the SP18 sequence, those residues are as illustrated in Figure 1.
In ~~ne embodiment, a subject polypeptide consists essentially of at least about 10, preferably at least 11 amino .acid residues, and no more than about 60, more usually fewer than about 35 and preferably fewer tlhan about 25 amino acid residues that correspond to the sequence of SP18 monomer.
Usually, the amino acid sequence of a polypeptide of this invention will correspond to a single group of contiguous residues in the linear sequence of SP18.
However, polypeptides that correspond to more than one portion of the SPlt3 sequence are also contemplated.
Usually at least one sequence that corresponds to at least 10, preferab:Ly at least 15, contiguous residues of the hydrophobic region of SP18 will be present in the peptide. A plurality of hydrophobic region amino acid sequences may be present.
A subject= polypeptide will preferably include as its carboxy terminal sequence at least 5 contiguous residues in t:he linear sequence of SP18 including residue 80. 'Thus t:he polypeptides of this invention may include o:ne or more groups of amino acid residues that correspond to portions of SP18 so that a sequence corresponding to a first group of contiguous residues ~3~) ~~0 of the SP18 monomer may be adjacent to a sequence corresponding to a second group of contiguous residues from the samE: or another portion of the SP18 monomer in the polypeptide sequence. Peptides having two or more sequences that correspond to a single group of contiguous amino acid residues from the linear sequence of ~~P18 is also contemplated.
Exemplar~~ preferred subject polypeptides corresponding in amino acid residue sequence to human SP18 monomer hydrophobic region are shown in Table 1.

~ 341 07 0 Designation) Amino Acid Residue Sequence pl-15 FPIfLPYCWLCRALI
pll-25 CRAhIKRIQAMIPKG
5 p21-35 MIPR:GALAVAVAQVC
p 31-4 5 VAQV'CRWP LVAGG I
p41-55 VAGGICQCLAERYSV
p46-76 CQCLAERYSVILLDTLLGRMLPQLVCRLVLR
p51-65 ERYSVILLDTLLGRM
10 p51-72 ERYSVILLDTLLGRMLPQLVCR

p54-72 SVILLDTLLGRMLPQLVCR
p54-76 SVILLDTLLGRMLPQLVCRLVLR
p61-75 LLGRMLPQLVCRLVL
1 The designation of each peptide indicates that portion ~~f they amino acid residue sequence of human SP18 monomer, as shown in Figure 1 to which the peptide sequence corresponds, i . a . , it indicates the location of the peptide sequence in the protein sequence .
In preferred embodiments, a subject polypeptide is further characterized as having a carboxy-terminal amino acid residue sequence represented b:y the formula:
-RLVLRCSMDDZ, wherein Z is an integer having a value of 0 or 1 such that when Z i;s 0 the D residue to which it is a subscript is absent: and when Z is 1 the D residue to which it is a subscript is present. Exemplary preferred "ca:rboxy-terminal polypeptides" are shown in Table 2.

~~41 070 Designation Amino Acid Residue Sequence p71-81 CRLVLRCSMDD
p66-81 LPQLVCRLVLRCSMDD
p59-81 DTLLGRMLPQLVCRLVLRCSMDD
p52-81 - RYSVILLDTLLGRMLPQLVCRLVLRCSMDD

p36-81 RWfLVAGGICQCLAERYSVILLDTLLGRMLPQLVCRLVLRCSMDD
p32-81 AQVCRWPLVAGGICQCLAERYSVILLDTLLGRMLPQLVCRLVLRCSMDD
1 The designation is the same as in Table 1.
Preferably, a subject polypeptide has an amino acid residue sequence that corresponds to a portion of the sequence shown in Figure 1. However, it should be understood that a polypeptide of the present invention :need not be identical to the amino acid residue sequence of a native SP18 monomer.
Therefore, a polypeptide of the present invention can be subject to various changes, such as insertions, deletions and substitutions, either conservative or non-conservative, where such changes provide for certain advantages in their use.
Conservative substitutions are those where one amino acid res:i.due is replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another such as between arginine and lysine, between glutamic and aspartic acids or between g~_utamine and asparagine and the like. The term "conservative substitution" also ~34~ 070 includes the use c~f a substituted amino acid in place of an unsubst:ituted parent amino acid provided that such a polypeptide also displays the requisite binding activity.
In one preferred embodiment, a serine (S) residue is substituted for a cysteine (C) residue, usually at lE:ast one of residue positions 71 and 77.
Preferably the serine analog has a sequence corresponding to the sequence of residues 51-76 of the SP18 monomer with the substitution at residue 71 or to the sequence of residues 51-81 with serine substitutions at 71 and 77.
When a pc>lypeptide of the present invention has a sequence that is not identical to the sequence of a native SP18 monomer because one or more conservative or non-conservative substitutions have been made, usually no more than about 20 number percent and more uaually no more than 10 number percent of the amino acid residues are substituted, except where additional residues have been added at either terminus as for the purpose of providing a "linker" by which t=he polypeptides of this invention can be conveniently affixed to a label or solid matrix, or carrier.. Labels, solid matrices and carriers that can be used with the polypeptides of this invention are described hereinbelow.
Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues that do not correspond in amino acid residue sequence to a native SP18 monomer.
Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. In addition, a polypeptide sequence of this invention can differ from the natural sequence by t:he sec;uence being modified by terminal-NHz acylation, e.g., acetylation, or thioglycolic acid amidation, terminal-carboxlyamidation, e.g., ammonia, methylamine, etc.
When coupled to a carrier via a linker to form what is known in the art as a carrier-hapten conjugate, a polypeptide of the present invention is capable of inducing antibodies that immunoreact with SP18 monomer. In view of the well established principle of immun.ologic cross-reactivity, the present invention thE~refore contemplates antigenically related variants of t:he polypeptides shown in Tables 1 and 2.
An "antigeni<:ally related variant" is a polypeptide that includes at least a six amino acid residue sequence portion of a polypeptide from Table 1 or Table 2 and which is capable of inducing antibody molecules that immunoreact with a polypeptide from Table 1 or 2 and an SP18 monomer.
In another embodiment, a polypeptide of this invention has amino acid residue sequence that has a composite hyctropho:bicity of less than zero, preferably less than or equal to -l, more preferably less than or equal to -2. Determination of the composite hydrophobicity value for a peptide is described in detail in Example :2. These hydrophobic polypeptides perform the function of the hydrophobic region of SP18. In a preferred embodiment, the amino acid sequence mimics the pattern of hydrophobic and hydrophilic residues of SP18.
A preferred hydrophobic polypeptide includes a sequence having alt=ernating hydrophobic and hydrophilic amino acid residue regions and is characterized as having at least 10 amino acid residues representE~d by the formula:
f aUb ~ c f d X341 07~

Z and U are amino acid residues such that at each occurrence Z and L;f are independently selected. Z is a hydrophilic amino acid residue, preferably selected from the group consisting of R, D, E and K. U is a hydrophobic <~mino acid residue, preferably selected from the group consisting of V, I, Z, C, Y and F.
"a", "b", "c" and "d" are numbers which indicate the number of hydrophilic or hydrophobic residues. "a"
has an average value of about 1 to about 5, preferably about 1 to about 3. "b" has an average value of about 3 to about 20, preferably about 3 to about 12, most preferably, about 3 to about 10. "c" is 1 to 10, preferably 2 to 10, most preferably 3 to 6. "d" is 1 to 3, preferably 1 to 2.
By stating that the amino acid residue represented by Z and U i~~ inde:pendently selected, it is meant that at each occurrence a residue from the specified group is selected. That is, when "a" is 2, for example, each of the h.ydrop:hilic residues represented by Z will be independently selected and thus can include RR, RD, RE, RK, DR, DD, DE, DK, etc. By stating that "a" and "b" have average values, it is meant that although the number of residues within the repeating sequence (Za Ub) can vary :somewhat within the peptide sequence, the average values of "'a" and "b" would be about 1 to about 5 and about :3 to about 20, respectively.
Exemplary preferred po:lypeptides of the above formula are shown in Table 3.

~ 34 ~ 07 0 Designation) Amino Acid Residue Seguence 1 The designation is an abbreviation for the indicated amino acid residue sequence.
Also contemplated are composite polypeptides of 10 to 60 amino aicd residues. A composite polypeptide consists essentially of an amino terminal sequence and a carboxy terminal sequence. The amino terminal sequence has an amino acid sequence of a hydrophobic region polype~ptide or a hydrophobic peptide of this invention, preferably hydrophobic polypeptide, as defined in the above formula. The carboxy terminal sequence has the amino acid residue sequence of a subject carboxy terminal peptide.
A polypeX>tide of the present invention can be synthesized by any techniques that are known to those skilled in the pol:ypeptide art. An excellent summary of the many techniques available may be found in J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969, and J. Meienhofer, "Ho_rmonal Proteins and Peptides", Vol.
2, p. 46, Academic Press (New York), 1983 for solid phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides", Vo:L. 1, Academic Press (New York), 1965 for classical solution synthesis.
In general, these methods comprise the sequential addition of one or more amino acid residues ~ X41 07 0 or suitably ~~rotecaed amino acid residues to a growing peptide chain. Ncrrmally, either the amino or carboxyl group of the first. amino acid residue is protected by a suitable, ;>elect.ively removable protecting group. A
different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
Using a solid phase synthesis as exemplary, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complimentary- (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from 'this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so fortla. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support;) are removed sequentially or concurrently, to a:Eford the final polypeptide.
H- Synthetic Surfactants Recombinantly produced SP18 and/or a subject polypeptide can be admixed with a pharmaceutically acceptable phospholipid to form a synthetic pulmonary surfactant (PS) useful in the treatment of respiratory distress syndrome.
The phase "pharmaceutically acceptable"
refers to molecular entities and compositions that do ~34~ 070 not produce an allergic or similar untoward reaction when administered to a human.
Phosphol.ipids useful in forming synthetic alveolar sur:Eactar,~ts by admixture with protein are well known in the art. See, Notter, et al, Clin.
Perinatology,, 14:433-79 (1987), for a review of the use of both native and synthetic phospholipids for synthetic surfactants .
In one embodiment, the present invention contemplates a synthetic pulmonary surfactant effective in treating RDS comprising an effective amount of a :subject polypeptide admixed with a pharmaceutically acceptable phospholipid. While methods for determining the optimal polypeptide:phospholipid weight ratios for a given polypeptide-F~hospholipid combination are well known, therapeutically effective ratios are in the range of about 1:5 to about 1:10,000, preferably about 1:100 to about 1:5,OOC, and more preferably about 1:500 to about 1:1000. In a more preferred embodiment, the polypeptide:phospholipid weight ratio is in the range of about 1:5 to about 1:2,000, preferably about 1:7 to about 1:1,000, and more preferably about 1:10 to about 1:100. Thus, a synthetic pulmonary surfactant of this invention can contain about 50, usually about 80, to almost 100 weight percent lipid and about 50, usually about 20, to less than 1 weight percent polypeptide.
Preferably a subje<a polypeptide is about 1 to about 10 weight percent of the surfactant for polypeptides corresponding to portions of the SPlB sequence and 1:100 for polypeptides corresponding to the entire SP18 monomer.
The lipid portion is preferably about 50 to about 90, more preferably about 50 to about 75, weight percent dipal:mitoy7Lphosphatidylcholine (DPPC) with the ~ 34T 070 remainder unsaturated plzosphatidyl choline, phosphatidyl glycerol (PC~), triacylg:Lycerols, palmitic acid sphingomylein or admixtures thereof:. Thc= synthetic pulmonary surfactant is prepared by admixing a solution of a subject polypeptide with a suspension of liposomes or by admixing the subject polypeptide and lipids directly in t=he presence of organic solvent. The solvent is then rE~moved by dialysis or evaporation under nitrogen and/or e~:posurEa to vacuum.
A subject synthetic pulmonary surfactant is preferably formulated for endotracheal administration, e.g., typically as a lic.uid suspension, as a dry powder "dust", or as an aerosol. For instance, a synthetic surfactant (polypeptide-lipid micelle) is suspended in a liquid with a pharmaceutically acceptable excipient such as water, saline, dextrose, glycerol and the like. A surfact:ant~-containing therapeutic composition can also contain small amounts of non-toxic auxiliary substances such as pH buffering agents including sodium acetate, sodium phosphate and the like. To prepare a synthetic surfactant in dust form, a synthetic surfactant is prepared as described herein, then lypoholized and recovered as a dry powder.
If it is to be used in aerosol administration, a subject synthetic surfactant is supplied in finely divided form along with an additional surfactant and propellent. Typical surfactants which may be administered are fatty acids and esters. However, it is preferred, in the present case, to utilize the other components of the surfactant complex DPPC and PG. Useful propellants are typically gases at ambient conditions, and ar~~ condensed under pressure. Lower alkane and fluorinated alkane, such as Freon*, may be used. The aerosol is packaged in a c~~ntainer equipped with a suitable valve so *Trade-mark ~34~ 070 that the ingredients may be maintained under pressure until released.
A synthetic surfactant is administered, as appropriate to the dosage form, by endotracheal tube, by aerosol administr~ition, or by nebulization of the suspension or dust into the inspired gas. Amounts of synthetic PS between about 1.0 and about 400 mg/kg, preferably about 50 mg to about 500 mg/kg, are administered in one dose. For used in newly born infants, one to three administrations are generally sufficient. For adults,, sufficient reconstituted complex is administered to produce <~ PO2 within the normal range (Hallman, et al, J. Clinical Inve:~tigation, 70:673-682, 1982).
The following examples are intended to illustrate, but not limit, the present invention.
Examples Example 1 - I~~olation and Characterization of Native SP18 A. Metr.oas Purification of LMW apoz~roteins Human pulmonary surfactant was isolated from full-term amniotic fluid and applied to a column of DEAE-Sephacel*
A-50 (Pharmacia, Uppsala, Sweden) using 4 milliliter (ml) packed volume per 200 milligram (mg) surfactant, in a tris-EDTA
buffer containing 1% n-octyl-beta-D-glucopyranoside as described by Revak, et a.l., Am. Rev. Respir. Dis., 134:1258-1265 (1986) and Hallman, et a.l., Pediatrics, 71:473-482 (1983). This particular column and conditions were used in order to isolate the 35,000 dalton apoprotein (for use in other studies) without exposing it to potentially denaturing organic solvents. The ,a' 30 void volume, containing the lipids and proteins which did not *Trade-mark ~34~ X70 bind to the column under these conditions, was pooled and extracted with an equal volume of 2:1 chloroform: methanol.
Following centrifugation to separate the phases, the upper phase (water + methanol) was re-extracted with 1/2 volume chloroform. After- centrifugation, the resultant lower organic phase was added to the :initial lower phase and evaporated to dryness under a stream o:f nitrogen. This extract, which contained 100-180 mg phospholipid, LMW apoproteins, and octylglucopyranosi.de, was redissolved in 2.5 ml of chloroform: methanol, 2:1.
Following the method of Takahashi, et al, Biochem.
Biophys. Res. Comrrl. , 135:527-532 (1986) , which was found to afford a good separation of octyglucopyranoside from the LMW
proteins and phos~~holipids, a glass column 2.5 cm in diameter was packed at 4° to a height of 38 cm with Sephadex LH-20 (Pharmacia, Uppsala, Sweden) in 2:1 chloroform:methanol. The sample was loaded and 2 ml fractions collected as chloroform:methancl, 2:1., was run through at a flow rate of 8.5 ml/hr. Phospholipids eluted after 40 ml of buffer had passed through the column. Octylglycopyranoside appeared at the 56-116 ml region.
The phospholi~>id region was pooled, dried under nitrogen, and redissolved in 1 ml chloroform. A silicic acid column was prepared by packing 9 ml of Bio-Sil HA* (BioRad, Richmond, CA) in chloroform in a glass column at room temperature. The sample (which contained approximately 50 mg phospholipid) was applied and washed with 11 ml chloroform. A
linear gradient of increasing methanol was established using an equal weight of chloroform and methanol (38.8 g, ~._.
~:~, ,;
Trade-mark ~~4~ 070 26.5 ml chloroform and 50 ml methanol). Fractions of 2 ml were co.llecte:d as the gradient was applied to the column. Figvure 1 shows the protein and phospholipid profiles obtained.
Phospho)_ipid analyses showed a small peak in fractions 17~-20 anal a major peak after fraction 30.
The Pierce Bc:A protein assay was positive in fractions 12-19 and 28--33, but it should be noted that the latter peak .Ls likely to be due to the phospholipid present in this region. Electrophoresis in sodium dodecyl sulfate polyacrylamide gels showed the LMW
apoproteins were present in fractions 13-19 with some separation occurring between SP9 and SP18.
Alternatively,, a method devised by Hawgood, et al, Proc. Natl. Acad. Sci., 85:66-70 (1987) employing a butanol extraction of PS followed by chromotography of Sephadex LH-c.0 in .an acidified chloroform/methanol buffer; can be used to isolate the LMW apoprotein mixture. For some studies, a separation of the two LMW apoproteins wars effected using Sephadex LH-60. A
glass column of 1 cm diameter was packed to 40 cm with Sephadex LH-60 (Pharmacia, Uppsala, Sweden) in chloroform/methano:L, 1:1, containing 5~ 0.1 N HC1. A
flow rate of 1-2 m:L/hr was used. A mixture of the LMW
apoproteins containing about 200-700 micrograms (ug) of protein from eit=her the Bio-Sil HA column or the LH-20 column described by Hawgood, et al, Proc. Natl.
Acad. Sci., 85:66-'10 (1987), in a volume of 0.5 ml buffer, was applied to the top of the column and fractions of 0.5 ml were collected. Typically, SP18 protein eluted in fractions 16-19 and SP9 in fractions 24-29. Appropriate fractions were pooled and dried in glass tubes under nitrogen. A brief period of lyophilization ensured complete removal of the HC1.
Proteins were re-solubilized in methanol prior to use.

SDS-Gel Electrophoresis Gel eleca rophoresis in 16$ polyacrylamide was performed in the presence of sodium dodecyl sulfate (SDS-PAGE) according to the method of Laemmli, Nature, 227:680-685 x;1970), using 3x7 cm minislab gels. 1$
~i-mercaptoeth.anol was added to samples where indicated as a disulfide reducing agent. Following electrophorescis, the gels were fixed overnight in 50$
methanol + lc.$ acetic acid, washed in water for 2 hours, and silver-stained according to the method of Wray, et al, Anal. Biochem., 118:197-203 (1981).
Octylglucop_yranoside Assay An assay for t:he guantitation of n-octyl-beta-D-glyco-pyranoside, based on the anthrone method of Spiro, Methods Enz~~mol., 8:3-5 (1966) has been described previously by Revak, et al, Am. Rev. Res ir.
Dis., 134:1258-1265 (1986).
Protein Determinations Organic samplea containing up to 5 ug protein were dried in 12x75 mm class tubes under nitrogen. Fifteen microliters (ul) of. 1$ SDS in H20 and 300 ul BCA
Protein Assay Reagent (Pierce Chemical Co., Rockford, IL) were admixed with the protein in each tube. Tubes were covered and incubated at 60°C for 30 min. After cooling, the :;amplea were transferred to a 96-well flat-bottom polystyrene microtiter plate and ODsso measured. Bo~,rine serum albumin was used as a standard. It should be noted that some phospholipids will react in the H~CA protein assay making protein quantitations inaccurate when lipid is present (i.e., prior to Bio-;;il HA. chromatography) . Additionally, once purified,. the hydrophobic LMW apoproteins ~34~ 470 themselves react poorly with the BCA reagents and all quantitations of t:he isolated proteins were, therefore, based on amino acid compositions.
Phospholipid~s Dipalmit:oylphosphatidylcholine (DPPC, beta, gamma-dipalmitoyl-:G-alpha-lecithin) and L-alpha-phosphatidyl~-DL-glycerol (PG, derivative of egg lecithin) were purchased from either Calbiochem-Behring (La ~Jolla, CA) or Avanti Polar-Lipids, Inc.
(Birmingham, AL). DPPC was added to PG in chloroform in a weight ratio of 3:1.
Admixture of LMW Apoproteins with Phospholipids For in vitro .assays, a methanol solution containing 4 ug of SP9 or SP18, was added to 400 ug DPPC:PG in chloroform in a 12x75 mm glass tube.
Following a brief vortex mixing, the samples were dried under rT2. Ninety microliters of water were added to each and t:he tubes placed in a 37°C water bath for 15 minutes, with periodic gentle mixing. Isotonicity was restored with the addition of 10 ul of 9$ NaCl to each sample prior t o assay. For in vivo rabbit studies, 50 ug LMW apoproteins (containing both SP9 and SP18) or 25 ug SP9 or 25 ug SP18 were dried under NZ. Five mg ~~f phospholipids (DPPC:PG, 3:1) were added in chloroform. The samples were mixed, dried, and resuspended in 250 ul 100 millimolar (mM) saline containing 1.5 mM CaCl2, to yield a reconstituted surfactant at 20 mc~/ml with 0.5-1$ protein.
Surfactant Activity Assays In vitro assays of surfactant activity, assessed as its ability to :Lower the surface tension of a pulsating bubble, and in vivo assays utilizing fetal rabbits, have both been described in detail previously by Revak, et al, Am._Rev. l~espir. Dis., 134:1258-1265 (1986).
Morphometric Anal~rses Fetal r~~bbit :Lungs, inflated to 30 cm H20 and then deflated to 10 cm H20, were submerged in 10% formalin for 72 hours. Paraffin :sections were oriented from apex to base and 5 micron sections taken anterior to posterior. After hematoxylin and eosin staining, 10 fields (100 x) were point-counted from apex to base on multiple sections. Standardized morphometric methods (Weiber, i.n "Stereological Methods," Vol. I, Academic Press, New York, pp. 33--58, 1979) were used to determine ratios of lung interstiti.um to air spaces for each treatment group.
Intersections of ~.lveolar perimeters were also determined.
Phospholipid Phos~~horus Assay Phospholipids were quantitated according to the method of Bartlett, J. Biol. Chem., 234:466-468 (1959).
Amino Acid Analysis Triplicate samples for amino acid compositions were hydrolyzed with HC1 at 1.10°C for 24 hours, with HC1 at 150°C
for 24 hours, or in performi.c acid at 110°C for 24 hours followed by HC1 hydrolysis at 110°C for 24 hours. Analyses were performed on a Beckman* model 121-M amino acid analyzer (Beckman Instruments, Fullerton, C"A). Tryptohan was not determined.
Amino Acid Sequenci~
Vapor-phase protein sequencing was performed on an Applied Biosystems* 470F. Amino Acid Sequencer (Applied ~~~:i:, ~ *Trade-mark 4 0 ' '~ '~ 1 0 ? 0 Biosystems, Inc., Foster City, CA) with an on-line Model 120A H:PLC.
Isolation of cDNA Clones for Human SP18 RNA was prepared according to Chirgwin, et al, Biochemistry,, 18:5294-5299 (1979) from a sample of unaffected adult lung tissue obtained during surgical removal of a neopl.astic lesion. Preparation of double stranded cDNA was carried out using standard techniques ((:hirgwin et al., supra, and Efstratiadis et al., in "Cienetic Engineering", eds. Stelow and Hollaender, F?lenum., New York, 1:15-49 (1979) and a library was constructed in lambda NM607 as described by Le Bouc, Ea al, F.E.B.S. Letts., 196:108-112 (1986). SP18 clones were identified by screening phage plaque: with synthetic oligonucleotide probes (Benton, et al, Science, 196:180-182 (1977) which were prepared using an .Applied Biosystems automated synthesizer and purified by HPLC. Initial candidate clones were abtain~ed using probe TG996 (5'CATTGCCTGTGGTATGGCCTGCCTCC 3') which was derived from the partial nucleotide sequence of a small human surfactant aP~oprotein cDNA (Schilling et al., International Patent Application WO 86/03408). Larger clones (up to 1.5 hb) were isolated using probe TG1103 (5'TCGAGCAGGA.TGACGGAGTAGCGCC 3') which was based on the 5' sequence of one of the original clones. The nucleotide sequencf~ of the cDNA clones was determined by the chain termination method (Sanger, et al, Proc.
Natl. Acad. Sci. U.S.A., 74:5463-5467 (1977) using EcoRI restriction fragments subcloned in an appropriate M13 vector.

B . Re~~ults Characteristics of: the LMW Apoproteins The LMW apoproteins isolated from human amniotic fluid appeared after silicic acid chromatography, or after the Sephadex: LH-20 column chromatography Figure 2 described by Haurgood, et al, Proc. Natl. Acad. Sci.
85:66-70 (1937), a.s two protein bands in SDS-polyacrylamic3e get electrophoresis under non-reducing conditions. The upper band, having an apparent molecular weight of 18,000 daltons is a dimer, and therefore designated SP18 dimer. With the addition of ~i-mercaptoetr~anol, SP18 dimer reduced to 9, 000 daltons ad was designated SP18 monomer (Fig. 3). The other LMW apoprotein, designated SP9, appears as a diffuse band between 9 and 12,000 daltons in the presence or absence of rE:ducing agents. SP9 was separated from SP18 dimer arid SP18 monomer by chromatography on Sephadex LH-E,O. 'The resultant purified proteins are shown in Fig. 3.
Amino acid compositions were determined for SP18 monomer and SP9. Because of the extremely hydrophobic nature of these proteins, HC1 hydrolysis was performed at 150°C for 24 hours, in addition to the standard 110°C for 24 hour hydrolysis, and values for valine, leucine, and isoleucine calculated from analyses of the hydrolysates done under the extreme conditions.
As shown in Table :3, both proteins are extremely hydrophobic with high levels of valine and leucine.

TABLE

Amino Acid Composition Human SP9 and SP18 monomer of and a Comparison with Theoretical Compos ition the of SP9 SP18 Splgl Amino Acid (mole (mole ~) (mole ~) Aspartic acid (or Asparag:ine) 1.1 3.4 3,7 Threonine 0.8 1.5 1.2 Serine 1.8 2.7 2.5 Glutamic acid (or Glutamine) 1.5 6.7 6.2 Proline 8.3 7.8 7.4 Glycine 10.6 6.1 4,g Alanine 4.9 10.2 g,g Cysteine2 9.1 7.2 g,6 Valine3 12.2 11.7 11.1 Methionine 3.4 3.2 3,7 Isoleucine3 6.8 6.4 7,5 Leucine3 22.4 17.4 17.3 Tyrosine 0.7 2.2 2.5 Phenylalanine 2.6 1.5 1.2 Histidine 5.4 0.0 0.0 Lysine 4.7 3.0 2.5 Arginine 3.9 9.0 8.6 Tryptophan N.D.' N.D.' 1.2 1 The theoretical composition is based on sequence data through residue 81.

2 Cysteine conte nt was g determined followin performic acid and HC1 hydrolyses.

3 Isoleuc:ine arid leucine content were each determined following 24 hour HC1 hydrolysis at 150°C' .
4 Tryptophan wa.s not determined.
Amino-terminal sequence analysis of SP18 monomer yielded the j:ollowing sequence:
NHz-Phe:-Pro-Ile-Pro-Leu-Pro-Try-.
Repeated sequencing of the purified SP9 monomer showed multiple peptides, all rich in leucine and containing at: least six consecutive valines. NHz-terminal analysis showed phenylalanine, glycine, and isoleucine with the relative amounts of each varying from preparation to preparation.
Nucleotide Se uenc~e Analysis of SP18 cDNA
The nucleotide sequence of a SP18 monomer cDNA
clone is presented in Figure 1. The sequence displays 83~ homology with ithe canine SP18 cDNA (Hawgood, et al, Proc. Natl. Acad. Sci., 85:66-70 (1987). A
sequence within a :Large open reading frame was identified which matches perfectly with the amino terminus of SP18 monomer as determined by Edman degradation of the isolated protein (underlined in Figure 3). This suggests that mature SP18 monomer arises by processing of a larger precursor molecule.
In the mature sequence there is a single potential N-glycosylation site (Asn 110), no sites for tyrosine sulfation, and no G-X-Y repeats as found in the 35,000 dalton apoprotein (White, et al, A. Pediatrics Research, 19:501-508; 1985).
The molecular weight of 9000 daltons obtained by SDS-PAGE of reduced SP18 dimer is lower than that predicted for the complete precursor protein sequence with amino terminus NH2-Phe-Pro-Ile-Pro-Leu-Pro-Tyr ~ 341 07 0 (19,772 daltons), implying further processing in the region of amino acids 70-90. In support of this, the theoretical amino acid composition (Table I) of a putative 900() dalton protein comprising residues 1 to 81 compares well with the determined values for purified SP18 monomer. The amino terminal portion of the precursor protein (residues 1 to 81) is alkaline and more hydrophobic than the COON terminal portion (residues 82 to 181): the Kyte-Doolittle index for residues 1 to 81 is 9100 (pI 8.6) and is -3000 (pI
5.91) for re~~idues 82 to 181 (Kyte, et al, _J.
Pediatrics, 1.00:619-622; 1982). The amino terminus (residues 1 t.o 81) is, as in the canine sequence (Hawgood, et al, P:roc. Natl. Acad. Sci., 85:66-70;
1987), composed of three hydrophobic domains: residues 1 to 11, 22 to 49 and 53 to 74. These are interspersed with a charged domain (residues 12 to 21) and two hydrophilic and charged stretches (residues 47 to 54 and 72 to 81;1.
Reconstitution of Surfactant Activity with LMW
Apoproteins Samples were prepared containing 400 ug/100 ul phospholipids (DPP(::PG, 3:1 by weight), phospholipids plus 4 ug SP9, or phospholipids plus 4 ug SP18. Each sample was assayed in the pulsating bubble surfactometer for t:he ability to lower surface tension. The results are shown in Table 4 as the mean minimal surface tension at 15 sec, 1 minute (min), and 5 min. Natural human surfactant, isolated from term amniotic fluid, diluted to 4 mg/ml is shown for comparison. 'v~hile neither phospholipids nor LMW
apoproteins alone had significant surface-tension lowering capa~~itie~~, a mixture of phospholipids with either SP9 or SP18 showed significant activity.

Recombining i~he phospholipids with 1~ by weight of SP18 lowered the surface tensions measured to levels comparable to those obtained with an equal amount of natural human surfactant (6.3 ~ 0.2 dynes/cm for 5 phospholipids + SP18 at 15 sec, 2.0 ~ 1.2 dynes/cm for natural surfactant). On an equal weight basis, SP9 lowered surface tension less effectively (16.7 ~ 0.8 dynes/cm at ~5 sec) .

Minimum Surface Tensions in the Pulsating Bubbles 15 sec 1 min 5 min PLZ 42.9 1.4 41.6 1.6 34.9 4.9 PL + SP93 16.7 0.8 14.1 1.2 12.2 1.0 15 PL + SP183 6.3 0.2 5.1 1.0 4.9 0.6 natural human surfactant' 2.0 1.2 2.4 1.4 0.4 0.4 1 Pulsation of 20 cycles/min started 10 sec 20 after bubble :formation. All values are in dynes~cm-~1 and are the average of at least 3 determinations.
2 Phospholipids DPPC:PG, 3:1, 4 mg/ml 3 1~ by weight compared to phospholipids 4 diluted to 4 mg/ml In vivo assay;; of exogenous (synthetic) surfactant activity were performed by instilling into the airways of immature fetal rabbits saline solutions containing Ca++ alone or with the addition of phospholipids, phospholipids plus LMW apoproteins, or natural human surfactant. The animals were ventilated for 30 min and then degassed by placement in a bell jar under vacuum. The lungs were then inflated to given pressures and the volume of air required for ~ 341 070 each pressurE~ was noted. The volumes required for given pressu:res during deflation from 30 cm HZO were likewise detE~rmine~d. The resulting pressure/volume curves are shown in Fig. 4 for animals which received synthetic surfactant made with purified SP9 or SP18 (0.5~ by weight compared with total phospholipid concentration) and appropriate control animals.
Improved lung compliance is apparent in those animals treated with natural or either synthetic surfactant as compared with those receiving saline or phospholipids with the SP18 appearing more effective than SP9 on an equal weight basis. A similar study was performed using a mixture of SP9 and SP18. The results were almost identical t~o the phospholipid plus SP18 curve presented in Fig. 4.
Following compliance measurements, the lungs were inflated to 30 cm HZO, deflated back to 10 cm HzO, clamped, excised and fixed in formalin. Thin sections were stained with lzematoxylin and eosin and examined microscopically, ids shown in Fig. 5, lungs treated with saline (A) or phospholipids (C) appeared atelectatic while i:hose from animals which received natural (B) or reconstituted (D) surfactant showed normal alveolar expansion. Morphometric analyses of the thin sections :>howed an interstitium to air space ratio of 4.70 for :>aline treatment and 3.29 for phospholipids alonE~ as compared with 0.498 for natural surfactant and 0.538 for reconstituted surfactant.
These data ar~~ shown in Table 5 and corroborate the significant (p<0.001; Mann-Whitney U Test) increase in air space seem in fig. 5. A comparison of alveolar perimeters similarly demonstrated a significantly (p<0.003) greater number of intersections of the alveolar bounciarie~; in saline- or phospholipid-treated fetuses compared to surfactant-treated animals.

Mo.rphomE:tric Analysis of Airspace F~~llowing Fetal Rabbit Treatment Tracheal Installation Interstitium/Air S ace saline 4.70 phospholipid~;l 3 . 2 9 phospholipid~l +
LMW Apoproteinsz 0.538 natural human surfactant3 0.498 1 2 mg of 3:1 Dl?PC:PG per animal 2 20 ug of LMW apoproteins added to phospholipids 3 2 mg per anim<31 C. Discussion This study describes two low molecular weight apoproteins isolated from human amniotic fluid surfactant which can be added to known phospholipids to produce a :biologically active pulmonary surfactant.
While the proteins in the current study have been designated as SP18 dimer, SP18 monomer and SP9, it is apparent from the recent literature that multiple nomenclature .and an assortment of reported molecular weights for LIYIW PS apoproteins (ranging from 5-18,000 daltons) exist. The apparent differences in physical properties ma:y be explained by a variety of factors including species differences, varying purification and handling i~echni.ques, varying determinations of low molecular weights based on standards in SDS-polyacrylamide gel~~, and potential interference by lipids of low molecular weight protein bands in gels.

~34~ ono Comparisons «f amino acid compositions and sequences and immunoloc~ic analyses using monospecific antibodies will help to sort out the LMW apoproteins.
It is felt that the SP9 protein described herein, giving a diffuse band on SDS-polyacrylamide gels from 9-12,000 daltons under reducing or non-reducing conditions, is probably the same protein as that designated SAP-6 by Whitsett, et al, Pediatric Research, 20:.744-749 (1986), SP5-8 by Hawgood, et al, Proc. Natl. Acad. Sci., 85:66-70 (1987), PSP-6 by Phelps, et a~_, Am. Rev. Respir. Dis., 135:1112-1117 (1987), and t:he 5 kDa proteolipid of Takahashi, et al, Biochem. Bio>hys Res. Comm., 135:527-532 (1986). The extremely hyctropho:bic nature of this protein is apparent from its .amino acid composition (Table 3) and sequence data., showing at least six consecutive valine residues preceded lby a leucine-rich region. The presence of three amino-terminal residues (phenylalanine, gl:ycine, and isoleucine) in the preparations of SP!~ derived herein from amniotic fluid surfactant suggest, a collection of peptides having an identical sequence but having had one or two residues removed from the amino-terminus. Phelps, et al, Am.
Rev. Respir. Dis., 135:1112-1117 (1987) have recently reported a similar finding with bovine PSP-6 apoprotein.
SP18 dimes is comprised of two identical 9000 dalton peptides (but different from the 9000 dalton peptide of SP9) that are disulfide linked. The amino acid composition of SP18 monomer (Table 3) shows a high number of hydrophobic residues. When unreduced SDS-PAGE were overloaded with SP18 protein, sequentially less intensely staining bands were seen at 36,000 and 56,00 0 daltons, suggesting oligomeric ~ X41 070 forms of the protean; upon reduction, only a single 9000 dalton J~and was seen.
Botl1 SP9 and SP18 dimer apoproteins isolated as described above, could be shown to have biophysical activity following recombination with phospholipids.
The addition of 1~ by weight of SP18 dimer to the phospholipids DPPf:PG resulted in an immediate increase in ;surface pressure resulting in surface tensions of ~_ess than 10 dynes/cm by 15 sec. The addition of ~_~ SP9 to DPPC:PG was slightly less effective, lowering surface tensions to 16.7, 14.1, and 12.2 dynE:s/cm at 15 sec, 1 and 5 min, respectively. Mixtures of both SP18 dimer and SP9 were also effective but further studies will be required to determine whether the combined effect is additive or ~:ynergistic.
In vivo studies of reconstituted surfactant using the fetal ralbbit model (Schneider, et al, J.
Pediatrics, 100:619-622; 1982) were performed using mixtures of SP18 d:imer and SP9 as well as each protein individually. A marked improvement in lung compliance was seen in animala treated with natural surfactant or reconstituted surfactant prepared with SP18 dimer apoprotein, as compared with those receiving phospholipids alone or saline (Fig. 4). A moderate improvement was seE~n when SP9 was used. Identical studies using a mi:~ture of SP18 dimer and SP9 to prepare the reconstituted surfactant showed results very similar to those obtained with SP18 dimer alone (solid squares, Fic~. 4); however, the exact ratio of SP18 dimer and SP9 in those studies could not be accurately ascertained. Fig. 5 shows representative microscopic alveolar fields indicating the lack of atelectasis following surfactant instillation.

Suz,iki, eat al, (Eur. J. Respir. Dis., 69:336-345; 1986) have reported a reduction in surface tension (meaaured on the Wilhelmy balance or in a pulsating bubble) and a five fold increase in tidal 5 volumes of prematurely-delivered rabbits at insufflation pressures of 25 cm and H20 when porcine LMW (<15,000 dalto~ns) surfactant apoproteins are added to mixtures of DPPC:PG) at a weight ratio of 5:80:20 (protein:DPPC:PG). Whether one or multiple proteins 10 are present ~_n this system is unclear.
Previous studies using the 35,000 dalton apoprotein (Revak, et al, Am. Rev. Respir. Dis., 134:1258-1265; 1986) also showed moderate reduction in surface tension, similar to that obtained with SP9 in 15 the studies ctescribed herein. Clearly, further studies must be done using various combinations and concentratior.~s of SP18, SP9 and the 35,000 dalton apoprotein, a:s well as Ca++ and perhaps various phospholipids: to elucidate the interactions between 20 these various components of surfactant and to determine the best conditions for a biologically active exogenous surfactant.
Example 2 - In Vitro Assessment, of Polype tide 25 Surfactant Activit~~
A. Methods Measurement of Sur:Eactant Activity Measurements of surface pressure across an air-liquid interface (Expressed in negative cm of HZO
30 pressure) at minimal bubble radius were determined at various times using the pulsating bubble technique described by Enhorning, J. Appl. Physiol., 43:198-203 (1977) .
Briefly, the F;nhorning Surfactometer 35 (Surfactometer International, Toronto, Ontario) measures the pressure gradient (~P) across a liquid-air interface of a bubble that pulsates at a rate of 20 cycles/min between a maximal (0.55 mm) a,nd minimal (0.4 mm) radius. The bubble, formed in a 37°C, water-enclosed, 20-ul sample chamber, is monitored through a microscopic o;pt:ic while the pressure changes are recorded on a stri~~ chart: recorder calibrated for 0 and -2 cm H20.
Determination of Composite Hydro hobicitv Value The compasite hydraphobicity value of each peptide was determined by assigning to each amino acid residue in a peptide its corresponding hydrophilicity value as described in Table 1 of Hopp, et al. Proc. Natl; Acad. Sci., U.S.A., 78:3824-3829 (1981).
For a given peptide, the hydrophilicity values were summed, the sum representing the composite hydraphobicity value.
Preparation of Synthetic Surfactants After admixture: with solvent, each peptide was combined with phospholipids (DFPt~~:PG)r 3:1 to form a synthetic surfactant according to one of the f:ollawing methods.
Method A was <~c~c°.omplished by admixing 16 ul of peptide/
solvent admixture (40 ug peptide) with 100 ul of chloroform con-taming 400 ug phospholip~i.ds, agitating the admixture for about 10 min. at 37°C to form a peptide/phospholipid admixture. Chloroform was removed from the pepti.de/phosphalipid admixture by drying under N2. The synthetic surfactant thus formed was then admixed with 90 ul of H20 and gen.t:ly agitated for about 10 minutes at 37°C. Subsequently, 10 ul. of ,, ~ ~: y, '~ i 9~ NaCl was admixed to the surfactant containing solution.
Method I3 was accomplished by first placing 100 ul of chloroform containing 400 ug of phospholipids in a glass tube and removing the chloroform by drying under NZ for about 10 minutes at 37°C . Sixteen ul of peptide/solvent admixture and 74 ul H20 were admixed with the driE:d phospholipids, and then gently agitated for about 10 minutes at 37°C. To the synthetic surfactant thus formed was admixed 10 ul of 9~k NaCl.
Method C; was accomplished by first maintaining the polypepti.de-PL admixture at 43°C for 10 minutes, after which time the solvents were removed from the admixture by drying under Nz. When needed, admixtures were further dried by 15 minutes exposure to vacuum to form a dried polyp~~ptide-PL admixture. Water was then admixed with each dried admixture in an amount calculated to equal 90~ of the volume necessary to give a final PL concentration of either 4 or 10 mg/ml (as indicated. in Table 7) to form a second admixture.
This second admixture was maintained for one hour at 43°C with agi~=ation. Subsequently, a volume of 6~ NaCl equal to 10~ of thES volume necessary to give the desired PL concentration was admixed with the second admixture and the resulting final admixture was maintained for 10 minutes at 43°C. In most cases, the final admixture was subjected to a last step of 3 cycles of freezing and thawing.
B. Results The synthetic surfactants illustrated in Table 6 were prepared as indicated in the table.

(2) (3) Phos- Composite (1) pholipid Hydro-Admixture Admixture phobicity PeptideSolvent Formed Method Value pl-15 n-propyl alcoholsuspens A -16.7 on pll-25 H,0 solution B + 1.7 p21-35 Chlorofoi:m suspension A _ 9.2 p31-45 HBO solution B - 9, g p91-55 H,O solution B - 5.4 p51-65 H,0 suspension B _ 2.2 p61-75 methanol suspension A _ g,g p71-81 H,0 suspension B + 3.9 p74-81 H,0 solution B + 3.7 p66-81 methanol:H20 suspension A - 1.0 p52-81 methanol:H,O suspension A _ 6.2 ( 1 ) Each p~~lypeF>tide was admixed with the indicated sc>lvent to achieve a concentration of 2.5 ug of peptide per ul of solvent .
(2) The lei~ters indicate the synthetic surfaci~ant F~reparation method used.
Those methodls are described above.
(3) The composite hydrophobicity value of each peptidE~ was determined as described above.
Each oj: the synthetic surfactants indicated in Table 6 were assayESd for surfactant activity as evidenced by their ability to reduce surface tension in vitro using the "bubble assay" of Enhorning as described above.
The re:~ults of this study, shown in Figure 6, indicate that a subject polypeptide, when admixed with pharmaceutically acceptable phospholipids, forms a synthetic pulmonary surfactant that has greater surfactant activity than the phospholipids alone, as evidenced by the lower AP values. Typically IO~S to 80~
lower DP values were obtained using the polypeptides.
It should be noted that the 8 amino acid residue control peptide p74-81, which does not conform to the teachings of the ~>resent invention, did not form a synthetic PS having a greater activity than the phospholipid alone:, thus indicating that amino acid residue lengt=h is a critical feature.
The surfact<3nt activity of additional exemplary polypeptides of this invention was studied using the "bubble assa~~" as described above. The results of the study are il_Lustrated below in Table 7.
Each polypeptide was admixed with the indicated solvent at a concentration of 2.5 mg of polypeptide per ml of so7_vent. The resulting admixture was observed to determine whether a solution or a suspension of: insoluble polypeptide was formed. Those admixtures forming a suspension were further admixed by water bath sonication for 10 seconds to form a very fine suspension, sufficient for pipetting using glass pipettes.
After <3dmixt.ure with solvent, each peptide was admixed with phosp:holipids (PL), DPPC:PG, 3:1, in chloroform in. a glass tube so that the amount of polypeptide added equaled one-tenth (10~ by weight) of the amount of PL added, to form a synthetic surfactant according to either method A, B or C.
Each oj° the synthetic surfactants was then assayed for surfactant activity as evidenced by their ability to reduce surface tension in vitro in the bubble assay performed as described above. The pressure gradient (dP) is a measure of surfactant activity in the polypeptide-PL final admixture which was determined using an Enhorning Surfactometer as described above. Measurements were obtained at time points of 15 seconds (15"), 1 minute (1') and 5 minutes (5') and are expressed as a mean of three independent measurE~ments of the indicated polypeptide-PL admixture. Pressure gradient measurements for comparable sa3mples~ of PL alone (PL) and natural human surfactants were determined as controls.
The results of this study are shown in Table 7.

(2) Phos- (3) (4) (1) pholipidConc. Pressure Admixture Admixtureof Gradient PL

Peptide Solvent-Formed Method mg/ml15" 1~ 5' -1 pl-15 N-propanol suspensionA 4 0.940.82 0.98 O

p36-81 50%chloroformsuspensionC+ 10 0.900.87 0.79 50%methanol p44-80 68%chloroformsolution C- 105 0 0 0 r 77 69 24 1 32%methanol . . .
J
' p96-76 64%chloroformsolution C+ 10 0.900.80 0.62 36%methanol 2 p51-72 75%chloroformsuspensionC+ 10 1.150.76 0.33 25%methanol p51-76 37%chloroformsolution C+ 10 0.990.91 0.92 63%methanol p51-80 95%chloroformsolution C+ 10 0.920.89 0.98 55%methanol p51-81 50%chloroformsuspensionC+ 10 0 0 0 3 50%methanol . . .

p52-81 67%DMF solution A 4 1.331.19 0.96 33%chloroform 3 p54-72 76%chloroformsuspensionC+ 10 1.280.92 0.38 24%methan~~l p54-76 71%chloroformsuspensionC+ 10 0.920.82 0.23 24%methanol p59-80 32%chloro:Eormsu;>pensionC- 105 1.100.96 0.69 68%methanol p59-81 68%chloro:Eormsolution C- 4 1 1 0 4 32%methanol . . .
Jr p69-80 98%chloro:Eormso.'LutionC- 105 1.070.87 0.11 52%methanol 5 p66-81 40%DMF su::pensionA 4 1.221.11 0.84 60%chloro,_'orm p74-81 water solution B 4 2.372.32 2.31 5 DL9 47%chloroi:ormsolution C- 9 2.001.80 1.30 (31 53~c methanol mer) RL9 32%chloroi:ormsolution C- 9 0.580.65 0.33 68%methanol RL8 19%chlorol:ormsuspensionC+ 10 0.680.69 0.19 81%methanol RRL7 49%chloroformsolution C- 9 1.651.25 1.00 1% methanol RCL-1 79%chloroi:ormsuspensionC+ 10 0.500.59 0.06 21%methanol 7 RCL-2 67%chloroformsuspensionC+ 10 0.000.00 0.00 33%methanol TABLE 7 - continued (2) Phos- (3) (4) (1) pholipid Conc. Pressure AcLnixture Admixture of PL Gradient Peptide Solvent - lEormed Method mg/ml 15" 1~ 5~
RCL-3 75'k chloro:Eorm suspension C+ 10 0.55 0.51 0.33 1 0 25% methanol PL C+ 10 >2.50 >2.50 2.33 Natural Human Surfactant 10 1.06 0.89 0.79 (1) Whether the initial admixture of peptide and was a solution or a suspension is indicated.
(2) The lett=ers indicate the synthetic surfactant preparation method used. Those methods are described above. A "+"
indicate s that the final admixture was subjected to a last step of 3 cycles of freezing and thawing. A "-" indicates the step wa:~ not performed.
(3) Concentration ("Conc.") of phospholipid (PL) in the final admixture is indicated in milligrams PL per milliliter admixture (mg/ml) .
(4) The pre~~sure gradient is a measure of surfactant activity in the polypeptide-PL
final actmixture as determined using an Enhorning Surfactometer as described in Example 2. Measurements were obtained at three points of 15 seconds (15"), 1 minute (1') and 5 minutes (5') and are expressed as a mean of 3 independent meaurements of the ndic:ated :polypeptide-PL admixture.
Pressure gradient measurements for compara~~le samples of PL alone (PL) and natural human surfactant are also shown.
(5) These solutions were made at a concentration of 20 mg/ml PL and were diluted to 10 mg/ml prior to testing.
These results indicate that a subject polypeptide, when admixed with pharmaceutically acceptable phospholipids,, forms a synthetic pulmonary surfactant that has a greater surfactant activity than the 1 341 07 p phospholipids alone, as demonstrated by the higher volume per g_LVen pressure.
Example 3 - In Vivo Assessment of Synthetic Surfactant Activity A ~ ME~thods Preparation of Synthetic Surfactants A subject polypeptide was first admixed with solvent as described in Example 2. The resulting admixture was further admixed with phospholipid (PL) so that the amount of polypeptide added was either 3%, 7% or 10% by weight of the amount of PL added as indicated below. The final polypeptide, PL admixture (synthetic surfactant) was formed according to method C using the final freeze thaw step as described in detail in they "Pre;paration of Synthetic Surfactants"
section in Example 2, except that the final admixture had a concentration of 20 mg phospholipid per ml of final admixture.
Instillation Protocols Protoco:L 1: Fetal rabbits were treated by injection into the trachea of a 0.1 ml solution that contained either a synthetic surfactant prepared in Example 3A or either 2 mg of native surfactant (NS) prepared as described in Example 1 or 2 mg PL.
Protoco=L 2: Synthetic surfactant was instilled in rabbit fetal lung by injection into the trachea from a single syringe of the following three components such that the components exit the syringe in the following order: (1) 0.05 ml air; (2) 0.1 ml of a synthetic surf=actant prepared in Example 3A or either 2 mg of PL or 2 mg of native surfactant; and (3) 0.1 ml air.

Protocol 3: From one syringe, a 0.1 ml aliquot of synthetic surfactant prepared as described in Example 3A (or 2 mg of NS or of Ph), was instilled into the rabbit trachea as described above, followed by injection of 0.05 ml lactated Ringer's Solution and 0.2 ml air from a second syringe.
Protocol 4: From one syringe, 0.1 ml of a synthetic sui:factant prepared as described in Example 3A (or 2 mg of NS or of PL), 0.15 ml air, 0.1 ml saline, and 0.3 ml air were injected into the trachea as described above. Two subsequent aliquots of 0.3 ml air were givE:n .
Protocol 5: Fetal rabbits were treated by injection into the trachea from a single syringe the following four components such that the four components exit the syringe upon injection in the order listed: (1) 0.2 ml solution that contains either a synthetic surfactant prepared in Example 3A
or either 4 mg of :native surfactant, or 4 mg PL; (2) a 0.15 ml volume of .air; (3) a 0.1 ml normal saline solution; and. (4) .a 0.3 ml volume of air. The above injection was then repeated 15 minutes after the first injection.
Protoco:L 6 P;abbits were treated as described in Protocol 5, except that two subsequent aliquots of 0.3 ml air were given :Following the initial instillation and no additional :instillation was given at 15 min.
Fetal Rabbit Model for Studying Surfactant Activit The surf=actant activity of exemplary polypeptides of this invention was studied using the methods described in detail previously by Revak, et al, Am.
Rev. Respir. Dis., 134:1258-1265 (1986), with the exceptions noted hE_reinbelow.

~~4~ 0~'0 Twenty-~>even day gestation fetal rabbits were delivered by hysterotom~r and immediately injected with 0.05 ml Norcuron* (Organon, Inc., NJ) to prevent spontaneous breathing.
The fetal rabbits were then weighed and a small cannula was inserted into the trachea by tracheotomy. Synthetic surfactant prepared as described above was then instilled into the fetal rabbit lung by one of the above instillation protocols.
Followir..g instillation the rabbit was placed in a specially designed. plethysmograph (containing a Celesco transducer) connected to a ventilator (Baby Bird, Baby Bird Corp., Palm Springs, CA) and the instilled lung was ventilated at a rate of 30 cycles per minute with a peak inspiratory pressure of 25 cm H20, a positive end expiratory pressure of 4 cm H20 and an inspiratory time of 0.5 seconds. In some studies, dynamic compliance measurements were made at various times throughout the ventilation procedure. In others, static compliance measurements were made following ventilation.
Static compliance measurements were made after 30 minutes of ventilation. The animals were removed from the ventilator and the lung; were degassed at -20 cm H20 in a bell jar under vacuum. Thereafter, the lungs were first inflated and then deflated through a T-connector attached to a tracheostomy tube. The volume of air required to reach static pressures of 5, 10, 15, 20, 25 and 30 cm H20 was measured during both inflation and deflation phases to generate static pressure/volume curves a.s a measure of static compliance.
Using the plethysmograph, dynamic compliance measurements were made at. various times throughout a 60 minute ventilation period. Corr~puter-assisted data *Trade-mark ~ 341 07 0 analysis resulted in compliance data expressed as ml of air per crn H20 per gram of body weight at each time point. Comp:Liance was calculated by the formula below.
Compliance = AV
AP
~Ptp - (C) -1, ~~V) + (R) , (F) Ptp - transpulmonary pressure C - compliance (elastic component - relates change in volume to pressure) R - resistance (relates flow to pressure) F - flow V - volume = the integral of flow with respect to time The above equation was solved with a multiple linear regression :Eor C and R. The compliance (C) represents the elastic nature of the lungs and the resistance (R) represents the pressure necessary to overcome the resistance to the flow of gas into and out of the lungs.
B. Results Static compliance data using instillation protocols 1 and 5 are shown in Figures 7 and 8, respectively. Improved lung compliance was seen in all lungs treated with natural surfactant or with the synthetic surfactants tested as compared with those lungs treated with phospholipids (PZ) alone, with one exception. T:he synthetic surfactant prepared using pl-15 (Figure 7) dial not produce improved lung compliance over PL alone when measured by static compliance.

~~~~o~o The results of the dynamic compliance studies are illustrat:ed in Table 8.

63 1 34 ~ ~7 0 Dynamic Compliance -n ml air/cm H,0 x 10 g body we ght Peptide Minutes after Sample Compared Surfactant Instillation Given By To PL 10 20 30 90 0 60 Protocol - - #

PL - - -_ 7 8 7 10 11 15 q 24 22 23 23 22 20 q 15 16 17 18 21 29 q NS -1 _ 265 251 168 186 173 197* q 918 388 905 288 237 * q 155 176 172 172 179 q 3p 0 5% 255 146* 3 5% 295 291 3 10% 154 1,162 2 10% 252 623 2 ~

10%' 27 87 138 207 323 6 10%' 20 23 35 59 87 136 6 5p 30 i 10%1 42 114 247 300 6 ' 10%

5p 5% 517 226* 3 5% 434 55* 3 10% 195 1,293 2 10% 43 1, 690 2 5p 10% 33 22 56 87 129 85 q 10% 10 11 186 358 141 149* q 10% 15 36 109 241 264 301 q 5p 4 5 10% 17 91 52 78 99 208 q 10% 76 94 149 199 217 308 q 10% 23 71 130 156 182 109* q 10% 92 114 247 388 6 5p 10%' 29 39 68 107 196 132 6 10%' 55 111 190 300 376 6 6p -55 10%' 63 129 235 318 6 P or to i these stillation into the ra bits, samples were filtered through 25 micron a filter.

60 * A decrea se compliance with time indicate in may the deve lopment of pneumothorax.

As shown in Table 8, each of the synthetic 65 surfactants oj: this invention and natural surfactant ~34~ p7p improved dynamic compliance values in comparison to phospholipid alone.
C. Discussion The in vivo compliance studies demonstrate that the use of a :number of exemplary synthetic surfactants of this invention resulted in enhanced compliance in comparison to phospholipid alone for each of the assayed synthetic surfactants. Thus, the proteins and polypeptides of this invention when admixed with pharmaceutically acceptable phospholipids form synthetic sur:Eactants that have greater surfactant activity than phospholipid alone. Use of the synthetic surfactants is advantageous in producing improved compliancE~ values in vivo.
Example 4 - St:udy of Binding of C-Terminal Peptide to Lung Epithelial Cells A. Meth~~ds Peptide Binding As:;~
A peptidE~ having residues 74-80 of SP18 (VLRCSMD) was radiolabeled by the Bolton-Hunter method (Bolton eta al, Biochem J., 133:529-538 1973) with l2sl (New England Nuclear - 34.1 moles/ml, 28.0 ng/ml, 75 ~,Ci/ml) .
Human pulmonary epithelial cells (human lung carcinoma cell, ATC;C reference no. CCL 185, commonly known as A549 cell~~) were grown to confluence in 6 well tissue c~ulture~ dishes. The following solutions were used in this ~;tudy:
PBS/BSA: 10 mM Na Phosphate + .15 M NaCl +
0.5$ BSA pH 7.4 Lysis Buffer: 1~ SDS in water Solution F: 5 ml PBS/BSA + 51.56 wg cold peptide Solution D: 2.5 ml PBS/BSA + 87 ~.1 l2sl-peptide Solution D1/5~: 0.5 ml D + 2.0 ml PBS/BSA

Solution D/125: 0.5 ml D1/5 + 2.0 ml PBS/BSA
Solution E: 2.5 ml PBS/BSA + 87 ~.1 l2sl-peptide +20.78 ~.g cold peptide Solution E1/5: 0.5 ml E + 2.0 ml PBS/BSA
Solution E 1/25: 0.5 ml E1/5 + 2.0 ml PBS/BSA
Three 6 well plates were pretreated by incubating with 0.5 ml of the following solutions for 15 min. at 22°C. The odd-numbered wells were pretreated with PBS/BSA and the even-numbered wells with solution F.
Following removal of the pretreatment solution, the wells were incubated with 0.5 ml of the following solutions at 22°C. for the indicated time while gently rocking the plates"
Well Samp:Le Incubation Time 1 D 7 minutes 2 E 7 minutes 3 D 1,~5 7 minutes 4 E 1,~5 7 minutes 5 D 1,125 7 minutes 6 E 1,125 7 minutes 7 D 30 minutes 8 E 30 minutes 9 D 1,~5 30 minutes 10 E 1/5 30 minutes 11 D 1/25 30 minutes 12 E 1/25 30 minutes 13 D 143 minutes 14 E 143 minutes 15 D 1~'5 143 minutes 16 E 1/5 143 minutes 17 D 1'25 143 minutes 18 E 1/25 143 minutes At the end of the incubation time the supernatant was removed from each well and saved for counting.
Each well way: washed four times with cold (4°C. ) PBS/BSA. The washes were saved for counting. The plate was then brought back to room temperature and 1 ml of lysis buffer was added to each well. The plate was gently shaken until all cells had lysed and come off the plate (3-4 minutes). The solution was removed from each well and counted. A second ml of lysis buffer was added to each well, mixed a few minutes and removed for counting of bound counts. The percent and absolute amounts o:E counts bound were determined.
Specific counts bound were determined by subtracting the counts bound in wells containing unlabeled (cold) pE~ptide from the corresponding well without cold peptide. The results are illustrated in Table 9, below.
The procedure was repeated with the following changes:
D1 = 1433 ~,1 PBS/BSA + 167 ~,1 l2sl-peptide [1.78 pmol/500 ~.1]
DZ = 183.3 Eil PBS/BSA + 366.7 ~,1 D1 [1.19 pmol/500 ~,1]
= 275 ~.l PBS/BSA + 275 wl D1 [0.89 pmol/500 ~.1]
D9 = 366.7 Eil PBS/BSA + 183.3 wl D1 [0.59 pmol/500 wl]
DS = 458.3 ail PBS/BSA + 91.7 ~,1 D1 [0.30 pmol/500 wl]
= 513.3 ~~l PBS/BSA + 36.7 ~,1 D1 [0.12 Pmol/500 ~,1]
E1 = 1386.24 ~,1 PBS/BSA + 167 ~,1 l2sl-peptide ( 4 . 676 ng] + 46.,76 ~,1 cold peptide at 100 ~g/ml [4.676 wg]
EZ-E6 Diluted as above for DZ - D6.
F - 3.398 ml PBS/BSA + 102.29 ~,1 cold peptide at 100 ~.g/ml.

~ 34 9 07 0 Two six-well plates were washed once with 1 ml PBS/BSA. They odd numbered-wells were pretreated with PBS/BSA and t:he even-numbered wells with solution F.
Following removal of the pretreatment solution, the following solutions were added.
Well Sample Well Sample 1 D~ 7 Da 2 E~ 8 Ea The solutions were incubated for 30 minutes at room temperature with gentle rocking. The supernatants were l.hen removed and saved for counting.
Each well was washed 4 times with 0.5 ml of cold PBS/BSA. Washes were saved for counting. 1 ml of 1~
SDS was added to each well to solubilize the cells.
After 3 minutes all the cells could be seen to have come off the plate., The lysed cell-containing supernatant was counted, together with a second SDS
wash of the wells. Total counts and the percentage of counts bound were determined. Specific binding was determined by subtz:acting the counts bound in wells containing cold peptide from the corresponding well without cold :peptide. The results are illustrated in Table 10.
B. Resu_Lts The resu_Lts of the binding studies are illustrated below in Tables 9 amd 10.

TABZE

Total CPM % Specific W
ll e Total CPM Bound Bound Difference Cts Bound 1 1,109,126 29,9142.23 2 1,087,659 17,3531.60 0.63% 6,930 1 0 3 223,170 4,479 2.01 9 221,608 9,l.la1.86 0.15% 330 5 45, 877 82f! 1.80 6 47,731 880 1.84 -0.09% - lg 7 1, 103, 25, 2.35 606 90'i 8 1,152,287 19,23(11.67 0.68% 7,980 9 227, 396 9, 2.20 2 0 1 99Ei 0 230,974 4,13i'1.79 0.91% 901 11 47,899 1,030 2.15 12 99,899 922' 1.85 0.30% 132 13 1, 151, 10, .87 19 1,108,755 9,50E~.86 0.01% 110 15 220,390 1,692 .77 16 229,253 1,800 .79 -0.02% - 49 17 46,893 907 .87 18 47,426 386 .81 0.06% 26 * Corrected to 100,000cpm/undiluted 1., tube.

4 CPM % Corrected ~

Well Total CPM Bound Bound Difference mol 1 3,0?0,705 66,959 2.78 2 2,995,775 56,055 1.87 9,390 1.78 3 2, 029, 39, 562 1.95 4 2,013,557 33,573 1.67 5,723 1.19 5 1,436,189 26,883 1.87 1,427,731 25,073 1.76 1,755 .g9 7 999,288 15,669 1.58 8 969,481 14,776 1.53 503 .59 9 460, 317 6, 513 1.41 10 479,746 6,816 1.42 - 52 .30 11 202,999 2,930 1.45 12 192,990 2,806 1.95 0 .12 * Corrected for 1.~7 -p mod , ,74 cpm C. Discussion As c;an be seen from the data in Table 9, the study demonstrated that the peptide was binding specifically to the cells as demonstrated by competitive inhibit ion by unlabeled peptide. However, the cells were not saturated by the amount of labeled peptide used in this study. Additionally, degradation of the peptide was occuring by 143 minutes.
The second study was performed using the 30 minute incubation period ;end an increased amount of labeled peptide to achieve saturation of the cells. As seen in Table 10, speci:Eic binding was again demonstrated.
Further, saturation was achieved as demonstrated by leveling-off of thE: amount of bound counts at high concentration of labeled peptide.
The binding studies thus demonstrate that the C-terminal peptide of this invention binds specifically to lung epithelial cells.
The foregoing specification, including the specific embodiments and examples, is intended to be illustrative of thE: present invention and is not to be taken as limiting. Numerous other variations and modifications can k>e effected without departing from the true spirit and scope of the present invention.

Claims (32)

1. A polypeptide consisting essentially of at least 10 amino acid residues and no more than about 60 amino acid residues, said polypeptide including a sequence having alternating hydrophobic and hydrophilic amino acid residue regions represented by the formula (Z a U b)c Z d, wherein Z is a hydrophilic amino acid residue independently selected from the group consisting of R, D, E and K;
U is a hydrophobic amino acid residue independently selected from the group consisting of V, I, L, C, Y and F;
a has an average value of about 1 to about 5;
b has an average value of about 3 to about 20;
c is 1 to 10; and d is 0 to 3;
said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.

2. The polypeptide of claim 1, wherein Z is independently selected from the group consisting of R and K.

3. The polypeptide of claim 1, wherein U is independently selected from the group consisting of V, I, L, C, and F.

4. The polypeptide of claim 1, wherein U is independently selected from the group consisting of L and C.

5. The polypeptide of claim 1 having an amino acid residue sequence selected from the group consisting of:
KLLLLKLLLLKLLLLKLLLLK, KLLLLLLLLKLLLLLLLLKLL, KKLLLLLLLKKLLLLLLLKKL, DLLLLDLLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLCLLLR, RLLLLCLLLRLLLLCLLLRLL, and RLLLLCLLLRLLLLCLLLRLLLLCLLLR.

6. A polypeptide having an amino acid residue sequence selected from the group consisting of:
KLLLLKLLLLKLLLLKLLLLK, KLLLLLLLLKLLLLLLLLKLL, KKLLLLLLLKKLLLLLLLKKL, DLLLLDLLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLCLLLR, RLLLLCLLLRLLLLCLLLRLL, and RLLLLCLLLRLLLLCLLLRLLLLCLLLR.

7. The polypeptide of claim 6, wherein said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forms a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.

8. A composite polypeptide of at least 10 and no more than 60 amino acid residues consisting essentially of an amino terminal sequence and a carboxy terminal sequence wherein:
said amino terminal sequence has at least 10 amino acid resides and has a composite hydrophobicity of less than 0; and said carboxy terminal sequence is characterized as consisting essentially of an amino acid residue sequence represented by the formula:
-RLVLRCSMDD Z, wherein Z is an integer having a value of 0 or 1, such that when Z is 0 the D residue to which it is a subscript is absent and when Z is 1 the D residue to which it is a subscript is present;
said composite polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.

9. The composite polypeptide of claim 8, wherein said amino terminal sequence is further characterized as having alternating hydrophobic and hydrophilic amino acid residue regions represented by the formula (Z a U b)c Z d, wherein:
Z is a hydrophilic amino acid residue independently selected from the group consisting of R, D, E and K;
U is a hydrophobic amino acid residue independently selected from the group consisting of V, I, L, C, Y and F;
a has an average value of about 1 to about 5;
b has an average value of about 3 to about 20;
c is 1 to 10; and d is 0 to 3.

10. A synthetic pulmonary surfactant comprising a pharmaceutically acceptable phospholipid admixed with a polypeptide according to any one of claims 1 to 9.

11. A synthetic pulmonary surfactant comprising one or more pharmaceutically acceptable phospholipids admixed with a therapeutically effective amount of a polypeptide consisting essentially of at least 10 amino acid residues and no more than about 60 amino acid residues, said polypeptide having an amino acid residue sequence that mimics the pattern of hydrophobic and hydrophilic amino acid residues of SP18.

12. A synthetic pulmonary surfactant comprising a pharmaceutically acceptable phospholipid admixed with a polypeptide having an amino acid residue sequence selected from the group consisting of:
KLLLLKLLLLKLLLLKLLLLK, KLLLLLLLLKLLLLLLLLKLL, KKLLLLLLLKKLLLLLLLKKL, DLLLLDLLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLCLLLR, RLLLLCLLLRLLLLCLLLRLL, and RLLLLCLLLRLLLLCLLLRLLLLCLLLR.

13. The synthetic pulmonary surfactant of claim 12, wherein said admixture has a surfactant activity greater than the surfactant activity of the phospholipid alone.

14. A synthetic pulmonary surfactant comprising one or more pharmaceutically acceptable phospholipids admixed with a polypeptide comprising at least 10 amino acid residues and no more than about 60 amino acid residues, said polypeptide including a sequence having alternating hydrophobic and hydrophilic amino acid residue regions represented by the formula (Z a U b)c Z d, wherein:
Z is a hydrophilic amino acid residue independently selected from the group consisting of R, D, E and K;
U is a hydrophobic amino acid residue independently selected from the group consisting of V, I, L, C, Y and F;
a has an average value of about 1 to about 5;

b has an average value of about 3 to about 20;
c is 1. to 10 ; and d is C to 3;
said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.

15. The surfactant of claim 14, wherein the value of d is 1.

16. The surfactant of claim 14, wherein the value of c is 4.

17. The surfactant of claim 14, wherein Z is a hydrophilic amino acid residue independently selected from the group consisting of R and K.

18. The surfactant of claim 14, wherein U is a hydrophobic amino acid residue independently selected from the group consisting of V, I, L, C and F.

19. The surfactant of claim 14, wherein U is a hydrophobic amino acid residue independently selected from the group consisting of L and C.

20. The surfactant of claim 14, wherein a is 1 or 2.

21. The surfactant of claim 14, wherein b has an average value of about 3 to about 9.

22. A synthetic pulmonary surfactant comprising one or more pharmaceutically acceptable phospholipids admixed with a polypeptide comprising at least 10 amino acid residues and no more than about 60 amino acid residues, said polypeptide including a sequence having alternating hydrophobic and hydrophilic amino acid residue regions represented by the formula (Z a U b) c Z d, wherein:
Z is a hydrophilic amino acid residue independently selected from the group consisting of R and K;

U is a hydrophobic amino acid residue independently selected from the group consisting of L and C;
a is 1 or 2;
b has an average value of about 3 to about 8;
c is 1 to 10; and d is 0 to 2;
said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.

23. The synthetic pulmonary surfactant of claim 14 or 22, wherein said polypeptide has an amino acid residue sequence selected from the group consisting of KLLLLKLLLLKLLLLKLLLLK, KLLLLLLLKLLLLLLLLKLL, KKLLLLLLKKLLLLLLLKKL, DLLLLDLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLCLLLR, RtLLLLCLLLRLLLLCLLLRLL, and RLLLLCLLLRLLLLCLLLRLLLLCLLLR.

24. A method of making a medicament useful for treating respiratory distress syndrome, comprising admixing one or more pharmaceutically acceptable phospholipids with a therapeutically effective amount of a peptide according to any one of claims 1 to 9.

25. Use of a therapeutically effective amount of a polypeptide according to any one of claims 1 to 9 to treat respiratory distress syndrome.

26. A commercial package comprising a therapeutically effective amount of a polypeptide according to any one of claims 1 to 9 together with instructions to treat respiratory distress syndrome.

27. Use of a therapeutically effective amount of a surfactant according to claim 10 to treat respiratory distress syndrome.

28. Use of a therapeutically effective amount of a surfactant according to any one of claims 11 to 22 to treat respiratory distress syndrome.

29. Use of a therapeutically effective amount of a surfactant according to claim 23 to treat respiratory distress syndrome.

30. A commercial package comprising a therapeutically effective amount of a surfactant according to claim 10 together with instructions to treat respiratory distress syndrome.

31. A commercial package comprising a therapeutically effective amount of a surfactant according to any one of claims 11 to 22 together with instructions to treat respiratory distress syndrome.

32. A commercial package comprising a therapeutically effective amount of a surfactant according to claim 23 together with instructions to treat respiratory distress syndrome.