Page 1
`
`RB Ex. 2009
`BDSI v. RB PHARMACEUTICALS LTD
`IPR2014-00325
`
`

`
`BUPRENORPHINE BIOAVAILABILITY
`
`50]
`
`distilled water and 10 ml BS 299. The extraction
`
`efficiency for [“H]buprenorphine was determined in
`triplicate by extracting control bloods each contain-
`ing an added quantity of labelled drug. The nature
`of the radioactivity in the extracts was checked for
`selected samples of each group of animals, after
`evaporation of the remaining organic extracts to
`low volume. The residues were chromatographed on
`silica t.l.c. plates (Merck silica gel 60 Fm) using a
`solvent system comprising ethyl acetate-methanol-
`NH.OH (sp.gr. 0-88), 75 :25 :1 (v/v). Authentic
`samples of non-radiolabelled buprenorphine were
`co-chromatographed with the extracts. Silica on the
`developed chromatograms was scraped into scintilla-
`tion vials as 1 cm bands then suspended in 2 ml
`distilled water and 5 ml ES 299 for analysis as
`coun‘ts min“ by liquid scintillation counting.
`Concentrations of buprenorphine in blood were
`obtained from the concentration of radioactivity in
`the extracts and corrected where necessary for both
`extraction efficiency and the relative proportion of
`buprenorphine in the extracts. Area under the curve
`values were calculated by trapezoidal analysis of the
`data.
`
`In situ absorption studies
`The absorption of PI-llbuprenorphine from the
`small
`intestine was measured using the in situ
`technique of Doluisio et al (1969). Male Sprague-
`Dawley rats (190-270 g) cannulated for bile collec-
`tion were each dosed intraduodenally with 12 ml
`(200 pg) of a [°H]buprenorphine solution prepared
`in 0-073 M sodium phosphate buffer, pH 6-5. The
`disappearance of radioactivity from the lumen was
`followed by measurement of suitable aliquots of the
`lumen contents at various times over the 90 min
`following dosing. The relative proportion of bupren-
`orphine in the lumen contents was determined by
`direct t.l.c. on silica plates as described earlier and
`from which it was then possible to calculate the
`actual percentage of buprenorphine still remaining
`in the gut lumen at any time.
`the small
`On termination of the experiment,
`intestine was removed, emptied, and then homogen-
`ized in 5 volumes of distilled water. Aliquots of
`the homogenates were combusted and assayed for
`radioactivity as described earlier. Samples of the gut
`homogenate were also treated with an equal volume
`of methanol, centrifuged and the supernatant com-
`position assayed by t.l.c. as described above. Bile
`was collected over the 90 min after dosing and total
`radioactivity determined by scintillation counting.
`Page 2
`
`
`
`-' units ml 1).
`_.
`- of the intravenous group was made by
`' into the left femoral vein whereas for the
`
`‘a! group the injection was made via an
`
`‘an to the heart.
`
`sublingual group the oesophagus of each
`‘
`_’7was ligated to prevent swallowing of the drug
`' animals dosed and maintained,
`in a face-
`.¢l‘» - ition. Intrahepatic portal vein and intra-
`1"": _l drug administrations were made by direct
`“ii. into the superior mesenteric vein and duo-
`Lrespectively.
`
`’ collection and assay
`samples (0-3 ml) were collected from the
`__.l cannula at suitable time intervals after
`§'=
`, the cannula being flushed with a solution
`its ml") of heparinized saline (0-3 ml) after
`issample. Bile was collected into weighed con-
`' each changed at 0-5, 1-0, 1'5, 2, 3 and 4 h
`Closing. The radioactivity in each bile sample
`"I" casured by counting duplicate aliquots (10 p.l)
`'i_ -
`I distilled water and 10 ml BS 299 scintillation
`-tail (Packard Instruments Ltd). Whole blood
`
`°ty (after drying over Ps0. for 3 days) by com-
`?_ on in 0, using a Packard 306 sample oxidizer.
`
`' r and d min"'1 values obtained using an on-line
`r interpolation quench curve program.
`'nchanged [°H]buprenorphine in whole blood
`-'3' determined by a differential extraction proce-
`'. . A measured aliquot of whole blood (100-200
`'_Was added to a glass extraction tube containing
`1“ pg non-radioactive buprenorphine carrier in
`_'_ -ml of 100 mM glycine/Na0l-I buffer pH 9-8. The
`,_-17 ..ples were then extracted with diethyl ether (3 X
`ml) and the organic extracts made up to 10 ml
`
`f- diethyl ether. Aliquots (3 ml) of each extract
`e evaporated to dryness and then counted in 1 ml
`
`cannulated towards the liver using pp 25
`tubing. To maintain patency of the
`trachea was exposed and fitted with a
`e cannula. The left carotid artery was
`"d cannulated towards the heart with a pp
`
`'
`
`'
`
`Page 2
`
`

`
`
`
`
`
`the curve (AUC) values up to 4 h after dosi 1:"
`each dose route. Taking the intraarterial
`represent complete (100%) bioavailability mg;
`tive availability of buprenorphine by tin".-5
`routes was intravenous (98°/0, rectal (547,), j I 1!
`hepatoportal (49%), sublingual (13%) and
`duodenal (9-7 %) (see Table l).
`
`"
`
`7 7
`Table 1. Relative bioavailabilities of buprenorphine
`.'
`various routes over the 0-4 h after dosing.
` F}
`
`-
`_
`Area under b_lood
`concentration Relative syste_m"I.‘
`time curve
`availability"(%) '
`(AUC ,,_.,,,, ng
`over the 0-4 11
`ml“ min)
`period
`
`Route
`
`I00‘
`1852 :1; 189
`Intraarterial
`98 3: 13
`1807 ;i; 242
`Intravenous
`54 :l: I4
`1000 i 267
`Intrarectal
`49 1 9
`900 i 161
`Intrahepatoportal
`13 5; 2
`249 :1:
`39
`Sublingualt
`9-7 _-J: 4
`180 ;§;
`71
`Intraduodenal
`
`
`=
`Z
`
`E
`1
`
`" Intraarterial route assigned to represent complete‘
`availability.
`1
`’rThe slow absorption profile for this route means
`any comparison of availability with other routes would‘
`be a considerable underestimate.
`Values represent the means of 4 animals zl: s.e.m.
`
`From the pharmacokinetic considerations des-I
`cribed by Cassidy & Houston (1980)
`it can be
`shown that
`the individual fractions (f) of (11118
`escaping clearance by the gut (fa), liver (fa) or lung
`(fn) are given by the expressions
`
`fa
`
`_ AUC (i.d.)
`AUC(h.p.v.)
`
`I
`
`AUC (h.p.v.)
`AUC (i.v.)
`
`L .
`
`: AUC (i.v.)
`AUC (i.a.)
`
`where AUC is the area under the curve to infinite
`
`time for the intraduodenal (i.d.), hepatic portal vein
`(h.p.v.),
`intravenous (i.v.) and intraarterial (i.a.}‘-
`routes. By reference to Fig.
`1
`it was clear that the
`0-4 h after dosing was insufficient in duration to‘
`permit an accurate determination of a terminal rate
`constant for the concentration curves and thereforfl
`
`AUCo_,,, values. However, since blood concentra-
`tions at 4 h after dosing were low and the curves
`(with the exception of sublingual) showed similar
`shaped profiles, fa,
`fix and fl. were calculated from.
`AUC,,_.,,, ratios. From these values the approximate
`first-pass clearance of buprenorphine by gut,
`liver
`
`Page 3
`
`502
`
`DAVID BREWSTER ET AL
`
`RESULTS
`
`Examination by t.l.c. of the ether extracts of all
`blood samples showed buprenorphine to be the
`major component
`(>7O"/0) and that
`this varied
`little between time of sample and dose route. The
`mean extraction efficiency of buprenorphine from
`control blood was shown to be 85 %.
`Blood concentrations of buprenorphine varied
`widely according to the route of administration, the
`intraarterial route providing the greatest availability
`and the intraduodenal the lowest (See Fig. 1). The
`
`10
`
`E0
`
`30
`
`
`
`Buprenorphineconcninblood(ngg‘)
`
`91‘
`
`01
`
`Time (h)
`
`I. Blood concentrations of buprenorphine in
`FIG.
`female rats following administration (200 pg kg '1) by
`various routes. Q, intraarterial; A,
`intravenous;
`rectal; A,
`intrahepatoportal; E].
`sublingual; I,
`intraduodenal. Points
`represent mean values of 4
`animals 3: s.e.m.
`
`relative bioavailability of buprenorphine by the
`different routes employed was determined for the 4 h
`period after dosing by calculation of the area under
`
`Page 3
`
`

`
`
`
`Fraction crossing tissue
`(f)
`
`AUC (i.d.)
`
`__
`
`_
`
`,
`
`_
`_
`__ AUC ('h.p.v.) __
`
`— -———-————AUC(ix)
`— 0 50 i 0 08
`
`_
`
`-.
`
`__ AUC (i.v.)
`1
`1, —
`
`_
`_
`__
`— 098 :l;013
`
`Mean
`first-pass
`effect (93)
`
`50
`
`2
`
`r- represents the area under the concentration
`e up to 4 h after dosing by the intraduodenal
`'trahepatoportal (h.p.v.),
`intravenous (1.v.) or
`‘al(i.a.) routes.
`for fa, fig, and f1. represent mean values :l:
`
`'1"!
`i
`
`lnistration of buprenorphine to rats was
`by a rapid excretion of drug-related
`_'l in the bile (See Fig. 2). However, biliary
`
`
`
`Time (h)
`
`.2. Cumulative biliary excretion of radioactivity by
`-'_
`.16 rats following administration of ["1-I] buprenor-
`C 200 pg kgr’ by various routes. Points represent
`__I_neans of 4 animals j: s.e.m. D, intrahepatoportal;
`: Intravenous; I, intraarterial; O, intrarectal; A,
`_ duodenal; V, sublingual.
`
`-i
`
`BUPRENORPI-UNE BIOAVAILABILITY
`
`503
`
`was calculated to be 80, 50 and 2%
`_‘_‘ly (Table 2).
`.irst-pass elimination of buprenorphine by gut,
`?-'lun8-
`
`-
`
`excretion after sublingual administration was rela-
`tively slow, presumably being dependent upon rate
`of mobilization of the drug from the sublingual and
`buccal cavities. The extent of biliary excretion over
`the 4 h varied with route of administration, being
`greatest after
`intrahepatoportal (mean 91%) and
`newest after intraduodenal (mean 46%) and sub-
`lingual (mean 22 %) administration. (Fig. 2).
`Buprenorphine appeared to be well absorbed from
`the small intestine as shown by the in situ absorption
`studies (see Fig. 3). For the first 30 min the radio-
`
`100
`
`100
`
`5 3o
`E
`2
`.5
`g
`--
`‘E1
`§ 10
`g
`‘D
`-‘°
`
`60
`
`
`30 c
`-2
`2
`S
`F.
`.9
`'5
`10%’
`2
`3
`3
`
`10
`
`30
`
`Time (min)
`
`50
`
`70
`
`FIG. 3. In situ absorption and subsequent biliary excre-
`tion of [‘H]buprenorphine in female rats. Insitu absorp-
`tion curve (0) represents the disappearance of bupren-
`orphine from the lumen. The biliary excretion curve
`(I) represents the cumulative excretion of total radio-
`activity in bile. All points represent
`the means of 4
`animals 1; s.e.m.
`
`activity in the lumen was almost exclusively un-
`changed buprenorphine, but after this time conju-
`gated drug began to appear as shown by t.l.c.-radio-
`assay of the lumen contents. The percentage of
`buprenorphine remaining in the lumen was therefore
`obtained by suitable correction of the total radio-
`activity data. Loss of buprenorphine from the lumen
`appeared to follow a biexponential decline (Fig. 3)
`with an initial phase showing a disappearance half-
`life of approximately 7 min and lasting until some
`
`Page 4
`
`Page 4
`
`

`
`504
`
`DAVID BREWSTER ET AL
`
`inter-animal
`siderable (Table 2) small
`result in relatively large variations in the --
`systemic availability. Generally, first-pass
`isms apply to the tissues gut, liver and lung, and __ _
`combination of metabolism by such tissues
`regarded as a first-pass effect.
`High metabolic clearance has been observe_t_;|_§u,_ -
`drugs such as lidocaine (Boyes et al 1970), proprufl-.i_ -_
`ol (Shand et al 1970) and salicylamine (Barr . 11
`i._-
`|[
`~1
`Of the more common drug delivery routes,
`administration is generally the most infiueneedh.-,_i
`first-pass elimination and this is because the gut .2
`liver effectively represent metabolizing systegfi '5
`arranged in series. Although metabolic activity is "
`gut has been known for many years (Hartiala 1973),.
`'
`the full metabolic capabilities of the tissue iiai;'i'_i:-
`until recently being underestimated. It is becoming‘
`apparent, however, that for some drugs this tissue
`of greater importance than the liver. Conway ettl
`(1973) for example, showed that intrahepatopoml
`or intraperitoneal administration of terbutaline to
`rats provided a 6 or 7 fold increase in availability-
`compared with the oral route, and Ilett et al (1980)
`have reported an extensive first-pass conjugation of
`isoprenaline in the dog intestine. It is apparent ako ,
`that phenolic opiates are extensively metabolized by
`gut tissue. In vitro studies have shown that buprenor-
`phine and other phenolic opiate agonists or antagon-
`ists undergo conjugation in rat gut and that the
`extent of conjugation is related to lipophilicilv
`(Rance & Shillingford 1977). These findings are
`supported by our observations that the first-pl!
`inactivation of buprenorphine during passage across
`the rat gut is of the order of 80 %. Morphine has also
`been shown to be extracted first-pass by rat gut and
`that the contribution of the gut was double that of
`the liver (Iwarnoto & Klaasen 1976). For phenol
`alone, a 94% first-pass extraction by rat gut has
`been shown to primarily account for an oral avail-
`ability in this species of only 33/;
`(Cassidy 31
`Houston 1980). Extraction of drugs by the gut can
`be minor compared with that of the liver, however. 35
`demonstrated by an increase in the oral bioavail-
`ability of lidocaine from 15 to 81% in dogs after
`surgical implantation of a portacaval shunt (Guglef
`et al 1975).
`
`
`
`_
`
`_
`
`'
`
`3
`
`'
`
`Rectal drug administration is preferred in some
`countries to the oral route and used in others when
`
`necessary as in patients with uncontrolled vomiting-
`Providing drug absorption from the rectum is
`efficient,
`rectal administration provides for im-
`proved delivery when oral drugs would otherwise
`suffer extensive pre-systemic extraction by the gut
`
`Page 5
`
`75% of the drug had been lost. A second phase
`accounting for the loss of remaining drug showed a
`half-life of some 15 min (Fig. 3)- The disappearance
`of buprenorphine from the gut
`lumen was con-
`sidered indicative of absorption of the drug since a
`considerable proportion of the original dose of‘
`radioactivity appeared in the bile of the same ani-
`mals (Fig. 3). Furthermore, assay of radioactivity in
`the gut wall after 10 min or 90 min of the in situ
`preparation showed a total content of only 20-28 "/0
`and 940% respectively of the original dose. Of the
`radioactivity recovered from the gut wall at these
`times, t.l.c. showed that a mean of 53 % (10 min) and
`73 % (90 min) was present as conjugated buprenor-
`phine, and is evidence of first-pass metabolic effects
`on buprenorphine in this tissue.
`
`DISCUSSION
`
`the
`is evident from the present studies that
`It
`systemic availability of buprenorphine is dependent
`upon the route of administration. For the 4 h after
`dosing, intravenous administration provided com-
`plete availability (98 %) relative to the intraarterial
`route. Delivery by the hepatic portal vein (49%) or
`rectal (54 %) routes was good, but characteristically
`for a phenolic opiate,
`intraduodenal availability
`(9-7 %) was low. Comparison with sublingual avail-
`ability (l3"/, over 4 h) is inappropriate since the
`blood profile was different from the above routes
`and appeared to be still in an absorption phase 4 h
`after dosing. Route dependence on the bioavail-
`ability of buprenorphine does not appear to be a
`consequence of variable absorption since the extent
`of biliary excretion over the 4 h was not propor-
`tional
`to drug availability. Furthermore,
`in situ
`absorption studies confirmed that buprenorphine is
`quickly and efficiently absorbed from the small
`intestine.
`
`Variability between delivery routw may often be
`a consequence of particular physicochemical proper-
`ties such that for example absorption from the gut
`is poor or that drug mobilization from a subcutane-
`ous injection site is incomplete. In the case of
`buprenorphine it is apparent that first-pass metabol-
`ism effects are the major influence. Pre-systemic
`elimination of drugs is becoming better understood,
`the number of documented examples is now con-
`siderable (Rowland 1977) and the pharmacokinetic
`consequences have been considered (Rowland 1972;
`Gibaldi & Feldman 1969). The first-pass effect on
`buprenorphine by gut and liver probably accounts
`for the inter-individual variation observed in the
`
`present studies. Since their first-pass efl‘ect is con-
`
`Page 5
`
`

`
`BUPRENORPHINB BIOAVAILABILITY
`
`S05
`
`C8VltY would not be expected to be as efficient as in
`a conscious man. It is predicted therefore that the
`overall availability of buprenorphine by the sub-
`lingual route is high and this has been confirmed in
`a recent clinical investigation (Bullingham et al 1981).
`Such bioavailability studies correlated with the
`eflicacy of sublingual buprenorphine in clinical
`studies (Edge et al 1979; Robbie 1979) and may be
`ascribed to the greater lipophilicity of the drug com-
`pared to morphine (Rance & Shillingford 1977),
`which by comparison is poorly and irregularly
`absorbed by this route (Edge et al 1979).
`There are important biological consequences
`which may result from the first-pass metabolism of
`drugs. After parenteral administration, buprenor-
`phine is extensively biliary excreted and an intestinal
`deconjugation and subsequent enterohepatic cycling
`of the drug occurs (Brewster et al 1981). However,
`this is slow and is not regarded to be of clinical
`significance because of first-pass inactivation during
`reabsorption. This may prove to be of greater
`importance after oral administration since the
`amount of drug recycled may be appreciable com-
`pared with the systemic availability by this route.
`Presystemic metabolism of buprenorphine occurs via
`conjugation at the 3-phenol group (Brewster et al
`1981) and does not appear to produce an active
`metabolite. The route dependence of the efficacy of
`buprenorphine is thus regarded as a consequence of
`inactivation rather than activation of the parent
`
`drug.
`
`REFERENCES
`
`Altman, G. E., Riseman, J. E. F., Koretsky, S. (1960)
`Am. J. Med. Sci. 240: 66-71
`Barr, W. H., Riegelman, S. (1970) J. Pharm. Sci. 59:
`164-168
`Boyes. R. N., Adams, H. J., Duce, B. R. (1970) J.
`Pharmacol. Exp. Ther. 174: 105-116
`Brewster, D., Humphrey, M. J., McLeavy, M. A. (1981)
`Xenobiotica 11: 189-196
`Brunk, S. F., Delle, M. (1974) Clin. Pharmacol. Ther.
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`Bullingham, R. E. S., McQuay, H. J., Dwyer, 1).,
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`Cassidy, M. K., Houston, J. B.
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`Conway, W. D., Singhvi, S. M., Gibaldi, M., Boyes.
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`Page 6
`
`
`
`'
`
`tcric vein to the portal vein, although the
`_I_j and middle haemorrhoidal veins connect
`_" y to the inferior vena cava. Studies in man
`lidocaine suggest
`the non-hepatic blood
`__-from the rectum to be around 50-60% (De
`_=_eta1 1979). Since in the rat, the liver does not
`the prime clearance organ for buprenorphine
`may not be a marked difference between man
`tin the systemic availability of buprenorphine
`' tered by the rectal route.
`most valuable non-invasive route of drug
`istration, which is also considered to bypass
`_ t and liver,
`is sublingual or buccal delivery
`-if
`review see Gibaldi & Kanig 1965). For drugs
`iflisuitable partition profiles, sublingual admini-
`can,
`in contrast to the oral route, provide
`.Vely rapid and efiicient drug delivery as
`plified by the use of glyceryl trinitrate in the
`"
`' nt of angina pectoris and isoprenaline in
`al asthma (Altman et al 1960). In the present
`__._t some evidence for the usefulness of the sub-
`..
`route for buprenorphine was seen. Although
`I:.1 bioavailability of the sublingually administered
`' was only marginally greater than when given
`..uodenally, it was clear from the plateau of
`11'
`I
`levels during the 1-4 h after dosing that drug
`_. still entering the systematic circulation long
`that by other routes. Furthermore, in contrast
`other routes, biliary excretion of drug-related
`(only 20% after 4 h) was still occurring
`_.;_T'.‘ almost
`linear manner 4h after sublingual
`.5118. In the anaesthetized animal model, blood
`and hence absorption from the sublingual
`
`_
`
`r. Certainly the value of the rectal route
`exemplified with drugs such as diazepam
`11 et al 1979) and lidocaine (De Boer et al
`. e present results with buprenorphine also
`value of the rectal route in the rat as
`by a five-fold increase in bioavailability
`oral route. However, the fact that rectal
`was still less than that by the intravenous
`" uld indicate a first-pass effect in the rectum
`__ h metabolism is a possibility since glucuron-
`erase activity is known to extend to lower
`:of the gut (Dawson & Bridges 1979). Trans-
`tal studies in animals to clinical potential
`f_ simple process because the relative propor-
`. 1’ blood drained systemically compared with
`_;_r-,_': ly probably varies between different species
`_
`. In rats, the evidence from studies with
`_' eand propranolol suggests blood return from
`'_'-‘- um to be mainly systemic. In man, however,
`
`Page 6
`
`

`
`506
`
`DAVID nnawsran ET AL
`
`Doluisio, J. J., Billups, N. 1=., Dittert, L. w. s
`
`'
`
`132.015., Swintosky, J. V. (1969) J. Pharm. Sci. 5'8: '1Jf$t6a—’
`1
`Edge, W. G., Cooper, G. M., M
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`Gibaldi, M., Kanig, J. L. (1965) J. Oral Thcr. Pharma-
`col. 1: 440-450
`
`, M,
`
`Gibaldi, M., Feldman, S. (1969) J. Pharm. Sci. 58:
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`
`Gugler, R., Lain, P., Azarnoff, D. L. (1975) J. Pharma-
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`
`Iwamoto, K., Klaasen, C. D. (1976) J. Pharmacol. Exp.
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`
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`
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`
`Magnussen, I., Oxlund, H. R. W,‘ Alsbirk K B.‘
`?7":‘9"6ds 5-
`
`Rowland, M. (1972) J. Pharm. Sci. 61: 70-74
`Rowland, M. (1977) in: Parke, D. v., Smith, R. L. -*'5}''
`Drug Metabolism from Microbe to Man. Taylor at‘) "
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`‘
`'-
`Shand, D. G., Nuckolls, E. M., Oates, J. A. (1970
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`-"
`Walle, T., Fagan, T. c., Conradi, E. c., Walle, u. 3''
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`(1979) Clin. Pharmacol. Ther. 26'
`7-172
`
`
`
`Page 7
`
`Page 7