United States Patent Office
`
`2,807,610
`Pa~ented Sept. 24, 1957
`
`2,307,6HI
`HYDROGENATED DEXTRAN
`.Mo~:ris Zief, E;iston, Pa., a~d Joseph R. Stevens, Asbury,
`N. J., assignors. to J. T. Baker Chemical Company,
`Phmipsburg, N. J., a cor_poratfon of New Jersey
`No Draw~ng. Applicaj,ion F,e~f!Iary 15, 195~,
`Serial -N"o· 271,!?44
`6 Claims. (Cl. 260~209)
`
`This invention relates to an -improved blood plasma sub-,
`stitute, which we have termed "clinica~ hydrodextran."
`Clinical dextran (degraded or hydrolyzed dextran) has
`been shown to be a promising blood plasma substitute in
`It is prepared by the
`quite extended· clinical studies.
`hydrolysis or splitting, usually acid hydrolysis, of native
`dextran, a high molf:cular weight fermentation product.
`In the hydrolytic or splitting procedure, the high molecu(cid:173)
`lar weight native dextran is degraded or split into mole(cid:173)
`cules of smaller size. The resulting product is fraction(cid:173)
`ated, as, for example, by addition of methyl alcohol to its
`·aqueous solution, whereby fractional precipitation is ef(cid:173)
`fected, the higher molecular weight products being pre(cid:173)
`dpitated at the lower •concentrations of alcohol. At the
`present time, molecular weight specifications for •clinical
`dextran are that the product have a weight average mo(cid:173)
`~ecular weight by light scattering of 75,000 plus or minus
`25,000 with the upper 5 to 10% having a weight average
`molecular weight ·not exceeding 200,000 •and the lower
`5 to 10% having a weight average molecular weight not
`below 25,000. As further investigation of the properties
`of dextran takes place, it may be that these specifications
`rfor clinical dextran will be changed, either in- the <;lirec(cid:173)
`tion of a :change in the weight average molecular weight,
`or further restriction on the upper and· lower Hmits or
`liberalization of the upper and lower limit_s.
`·
`Clinical dextran has been observed from time to time
`to cause undesirable side reactions. . ·
`· ·
`The product of the present invention, clinical hydro(cid:173)
`dextran, has substantially the same advantageous pmp(cid:173)
`erties of dextran, but differs from dextran ·in having a
`considerably lessened tendency to cause side reactions after
`injection. The product of the present inven~on is ob(cid:173)
`tained by subjecting cli.nical dextran to hydrogenation(cid:173)
`catalytic, electrolytic, or chemical-with conversion of
`the ·carbonyl groups to hydroxyl groups. The extent of
`hydrogenation is exceedingly small. Thus, with acid hy(cid:173)
`drolyzed or degraded dextran we have found that ab(cid:173)
`sorption of about one mole of hydrogen per mole con(cid:173)
`verts the dextran to the hydrodextran of the invention.
`This corresponds approximately to an increase in mo(cid:173)
`lecular weight from say 60,000 to 60,002, an amount
`much too small to be determined by any available method
`of determining molecular weight, the margin for error, for
`example, in determining weight average molecular weight
`of dextran by light scattering being of the order of 10%.
`While no analytic methods are available to enable us
`to determine exactly what the nature of the change in
`structure of the dextran is upon hydrogenation, we believe
`that it involves the reduction of carbonyl groups, which
`may be keto but probably are aldehyde (or hemi-acetal)
`groups in terminal positions to the ·corresponding hydroxyl
`groups, without hydrogenolysis or other reactions taking
`place. Evidence of this is the fact dinical dextran is
`reducing to the Somogyi reagent, whereas the product of
`the present invention is not. This definitely indicates the
`presence in the dextran of reducing carbonyl groups, which
`
`ti
`
`2
`are probably aldehyde groups, but may 'be keto groups
`resulting · from rearrangement of cyclic intermediates
`formed during the hydrolysis of the dextran when its acid
`solutions are heate.d for considerable periods of time.
`Dextran i.s sterilized by autoclaving. The final product
`is required to have a pH betwe.en 5 and 7, and in some
`1cases it is desirable to have the medium in which the
`. dextran is dissolved (isotonic saline) slightly alkaline prior
`to autoclaving because a tendency for the pH to drop dur-
`IO ing autoclaving is sometimes noted. Slight errors in ad(cid:173)
`justment of the pH lead to significant development of color
`and acidity. The product of the invention lacks this sen(cid:173)
`sitivity and hence is more easily sterilized by autoclaving.
`The invention will be illustrated by the following spe(cid:173)
`; 5 cific examples but it is not limited thereto.
`Example I
`To 16.6 cc. of a .6 percent aqueous solution of clinical
`dextran (weight average molecular weight ·60,000 by Jight
`20 scattering) at 0-5° C. were added 0.5 N sulfuric acid and
`. 2 g. portions of 2 percent sodium amalgam at such a
`rate that the reaction medium was slightly acidic at all
`times. A total of 37 cc. of ·acid and 22 g. of sodium
`amalgam was added. After removal of the mercury, the
`25 solution was neutralized, deionized by passage through
`cation (IR-100) and anion (IR:_4B) exchange resins, and
`added to methyi alcohol with stirring ( 1 part solution to
`9 parts methyl aicohol). The ·resulting precipitate was
`sep!lrat!!d by centrifuging at 2,000 R. P. M. for 20 minutes,
`30 then powdered and dried in vacuo. The product was non(cid:173)
`rnd11cing to boiling Somogyi solution. The relative vis(cid:173)
`cosity of a 6 percent solution of the product at 25° C.
`was 3.41; the reiaiive viscosity of the original dextran was
`.
`- .•
`-3.45.
`.
`Example II
`A 20 cc. portion of 6 percent aqueous solution of clini(cid:173)
`cal dextran (weight average molecular weight 60,000 by
`light scattering) was shaken with approximately 0.4 g.
`40 of a 5 ,Percent paliadium on carbon catalyst under 54
`pounds of hydrogen pressure at 25° C. fOT 18 hours.
`After filtering· off the catalyst, the product was precipi(cid:173)
`tated from methyl alcohol as in Example I. The product
`was 1completely _non-redw;ing to Somogyi reagent. The
`45 relative viscosity of a 6 percent solution of the product
`was 3.45; the relative viscosity of the starting material was
`approximately the s.ame.
`Example III
`To a 10 percent aqueous solution containing 10 pounds
`of clinical dextl'an (weight average molecular weight
`60,000 by light scattering) were added 14 g. of sodium
`borohydride in 500 cc. of water. The mixture was al(cid:173)
`lowed to stand at mom temperature for 5 hours with oc-
`55 casional stirring and was then acidified with 30 percent
`acetic acid. The acidified mixture was passed through a
`column of ·a cation ex<change resin (Amberlite IR~lOO)
`and the effluent was passed through a column of an anion
`exchange resin (Amberlite IR-4B). Methyl alcohol was
`60 added with stirring to the de-ionized solution to give a
`solution 60 percent methyl alcohol by volume. After
`standing for 24 hours at 25° C., the supernatant solution
`was decanted from the precipitated reduced dextran. The
`product was dried at 100° C. at atmosphedc pressure for
`65 1 hour, then at 100° C. in vacuo for 2 hours. The prod(cid:173)
`uct was non-reducing to Somogyi reagent. The specific
`rotation [cxJD210
`, was +196.6 degrees, the relative viscosity
`of a 6 percent solution at 25° C. was 3.42, and the in(cid:173)
`trinsic viscosity at 25° was 0.21 dl./g. The product was
`70 boron-free and the weight average molecular weight by
`light scattering was 55,000.
`In this procedure the amount
`of sodium borohydride was twenty times the theoretical
`
`35
`
`50
`
`Luitpold Pharmaceuticals, Inc., Ex. 2016, p. 1
`Pharmacosmos A/S v. Luitpold Pharmaceuticals, Inc., IPR2015-01495
`
`

`
`2,807,610
`
`3
`quanti.ty required for reduction of all carbonyl groups,
`assum~ng an average molecular weight of 60,000, and
`assummg one carbonyl, probably aldehyde group per
`molecule.. Results when other proportions of sodium
`borohydride were employed are given in the following
`table.
`
`Reac(cid:173)
`tion
`time
`in
`hours
`
`Intrin(cid:173)
`sic vis(cid:173)
`cosity
`(di.Jg.)
`
`Rel.
`vise.,
`63
`solu.,
`25° c.
`
`NaBHj
`ratio: R_cdnc-
`actnal mg
`amt. to power 1
`theory
`
`No. L------------
`No. 2 _____________
`No.3 _____________
`Clinical dextran
`(mo. wt.,
`60,000) __________
`
`2. 5
`5.0
`4.0
`
`+rn1.0°
`+196. 6°
`+198.0°
`
`0.21
`o. 21
`0.21
`
`3.40
`3.39
`3.40
`
`1, 100
`20
`2.5
`
`--------
`
`+199. 0°
`
`0. 21
`
`3.40
`
`0
`0
`98
`
`100
`
`30
`
`We have also considered the possibility that the de(cid:173)
`graded or hydrolyzed dextrans, as a consequence of the
`rel~tively drastic hydrolysis, contain carboxyl groups, and
`to msure complete absence of such reactive groups as
`5 carboxyl gn;ups from the final product, treated it with
`r~agents which remove any carboxyl-containing impuri(cid:173)
`ties. Thus, we have treated a warm alkaline solution of
`h~drodextran, pro~uced in accordance with Example III,
`with 1 % of alummum hydroxide, followed by filtration.
`10 ~his procedure removes any carboxyl-containing impuri-
`ties. To remove any traces of aluminum we have passed
`the clear filtrate through an ion exchange resin. This
`procedure gives a clinical product, hydrodextran, free from
`carbonyl and carboxyl groups.
`15 We claim:
`1. The method which comprises hydrogenating clini(cid:173)
`cal degraded dextran which is reducing to the Somogyi
`reagent until the degraded dextran absorbs sufficient hy(cid:173)
`drogen to become substantially non-reducing
`to . the
`20 Somogyi reagent.
`2. The method of claim 1 where the clinical degraded
`dextrai: has a weight average molecular weight by light
`scattermg of 75,000 plus or minus 25,000 with the upper
`5 to 1?% having a weight average molecular weight not
`25 exceeding 200,000 and the lower 5 to 10% havin" a
`weight average molecular weight not below 25,000. "
`3. The new product, hydrogenated clinical degraded
`de~tran, said degraded dextran prior to hydrogenation
`bemg characterized by being reducing to the Somogyi re(cid:173)
`agent and after hydrogenation being characterized by be(cid:173)
`ing substantially non-reducing to the Somogyi reagent.
`4. The new product, hydrogenated clinical de"raded
`dextran, said degraded dextran having a weight a~era"e
`m?lecular weight by light scattering of 75,000 plus ~r
`mmus 25,000 with the upper 5 to 10% having a wei"ht
`average molecular weight not exceeding 200,000 and fue
`lower 5 to 10% having a weight average molecular weight
`not below 25,000, said hydrogenated degraded dextran
`being substantially non-reducing to the Somogyi reagent.
`5. As a new product, hydrogenated clinical dc"radcd
`dextran, said product being substantially non-reduclng to
`the Somogyi reagent.
`6. Hydrogenated dextran consisting of a mixture of
`45 dextrans of v~rying molecular weights, the aldehyde end
`groups of which have substantially all been reduced to
`alcohol groups.
`
`1 Expressed as mg. of glucose per 500 cc. of 6% solution.
`In Example 3 the rate of reduction with sodium boro(cid:173)
`hydride was followed by titration. A 2 cc. sample was
`removed at hourly intervals, acidified with acetic acid to
`destroy excess sodium borohydride, then boiled with
`It
`Somogyi reagent and titrated with 0.005 N thiosulfate.
`was found that reduction was complete in 3 hours.
`Native dextran may be converted to a hydrodextran
`by a similar procedure, but we know of no advantage in
`following this practice because the reducing power of
`clinical dextran is very considerably greater than ·can be
`accounted for by the presence of the reducing groups in
`the native dextran, that is, it appears that they are formed
`in part at least, in the course of the degradation of th;
`native dextran to a molecular size appropriate for clinical
`use.
`The clinical dextran referred to in the three examples
`above was in each case a product obtained by subjecting
`native dextran to hydrolysis in approximately 0.1 N hydro(cid:173)
`chloric acid solution at temperatures between 90 to 100°
`C. Other methods of hydrolysis or degrading dextran
`have been suggested, for example, the use of ultrasonic
`vibrations. Products so obtained can also be readily
`converted to products of this invention by subjecting them
`to hydrogenation, the essential requirement being the ab(cid:173)
`sorption of sufficient hydrogen to reduce the reducing
`power against Somogyi reagent to zero or very close to
`it.
`We have not, in the hydrogenations we have canied
`out, cacountered any difficulties from hydrogenolysis or
`In general, the products have the same molecu-
`the like.
`lar weight as the starting materials, their solutions have 50
`the same viscosities ·and the other physical properties re(cid:173)
`main about the same.
`
`35
`
`40
`
`References Cited in the file of this patent
`UNITED STATES PATENTS
`Lautenschlager et al. _____ June 27, 1933
`Hartstra et al. ------------Aug. 8, 1950
`
`1,915,431
`2,518,235
`
`Luitpold Pharmaceuticals, Inc., Ex. 2016, p. 2
`Pharmacosmos A/S v. Luitpold Pharmaceuticals, Inc., IPR2015-01495