_
`
`UllltBd States Patent [19]
`Calne
`
`llllllllllllllIllllllllllllIllllIlllllllllIllllllllllllllllllllllllllllllll
`[11] Patent Number:
`5,100,899
`[45] Date of Patent:
`Mar. 31, 1992
`
`USOO5100899A
`
`[54] METHODS OFINHIBITING TRANSPLANT
`REJECTION IN MAMMALS USING
`RAPAMYCIN AND DERIVATIVES AND
`PRODRUGS THEREOF
`
`[76]
`
`[21]
`[22]
`[511
`[52]
`[53]
`[56]
`
`Inventor:
`
`Sir R. Calne, 22 Arrow Road,
`Cambridge, England, CB22AS
`Appl. No.: 362,354
`Filed:
`Jun. 6, 1989
`
`Int. Cl.5 ............................................ .. A61K 31/44
`U.S. Cl. ........................................ .. 514/291
`Field of Search ....................... .. 424/122; 514/291
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3,929,992 12/1975 Sehgal et al. ..................... .. 424/122
`3,993,749 11/1976 Sehgal et al. ..................... .. 424/122
`4.315688 2/1982 Rakhit ............................... .. 424/122
`4,650,803 3/1987 Stella et a1. ....................... .. 514/291
`
`OTHER PUBLICATIONS
`Chemical Abstracts ll2z353y (1990).
`
`Martel, R. R., et al., Can. J. Physio. Pharmacol.
`55:48-51 (1977).
`Staruch, M. 1., et al., FASEB. .1. 3(3):34l1 (1989).
`Dumont. FASEB Journal, vol. 3, #5256.
`Primary Examiner-—Jerome D. Goldberg
`Attorney, Agent. or Firm-Darby & Darby
`[57]
`ABSTRACT
`This invention provides a method of inhibiting organ or
`tissue transplant rejection in a mammal in need thereof,
`comprising administering to said mammal a transplant
`rejection inhibiting amount of rapamycin. Also dis
`closed is a method of inhibiting organ or tissue trans
`plant rejection in a mammal in need thereof, comprising
`administering to said mammal (a) an amount of rapamy
`cin in combination with (b) an amount of one or more
`other chemotherapeutic agents for inhibiting transplant
`rejection, e.g., azathioprine, corticosteroids, cyclospo
`rin and FK506, said amounts of (a) and (b) together
`being effective to inhibit transplant rejection and to
`maintain inhibition of transplant rejection.
`
`7 Claims, No Drawings
`
`Ex. 1070-0001
`
`

`
`1
`
`5,100,899
`
`METHODS OF INHIBITING TRANSPLANT
`REJECI'ION IN MAMMALS USING RAPAMYCIN
`AND DERIVATIVES AND PRODRUGS THEREOF
`
`FIELD OF THE INVENTION
`This invention relates to methods of inhibiting organ
`or tissue transplant rejection in mammals. More particu
`larly, the invention relates to methods of inhibiting
`transplant rejection in mammals in need thereof, com
`prising administering to such mammals a transplant
`rejection inhibiting amount of rapamycin.
`
`25
`
`35
`
`BACKGROUND OF THE INVENTION
`Rejection and infective complications resulting from
`immunosuppressive treatment are the principal causes
`of failure of organ allografting in man, that is, an organ
`graft made between two genetically different individu
`als in the same Homo sapiens species. In order to mini
`mize the individual speci?c side-effects of the three
`effective agents used in clinical practice, namely azathi
`oprine, corticosteroids and cyclosporin, small doses of
`each are used in combination “triple therapy". Of the
`three agents currently used in such triple therapy, cy
`closporin is the most powerful, but has the unsatisfac
`tory side-effect of nephrotoxicity in man which can lead
`to structural renal damage. Increased corticosteroid
`dosage and antilymphocyte antibody preparations,
`poly- or monoclonal, are used for the treatment of rejec
`tion crises. A number of studies have been taken to
`investigate other potentially effective compounds for
`use as immunosuppressants and transplant rejection
`inhibitors, but to date, none have been found to be use
`ful in clinical settings because of side-effects, such as
`toxicity, the lack of ef?cacy or a combination of these
`factors.
`The fungal product FK506 was reported to have
`immunosuppressive activity in animals with organ
`grafts (Ochiai, T., et al., Transplant. Proc, Vol. XX, No.
`1, pp. 209-214, 1988). Although the immunosuppressive
`activity of FK506 was con?rmed, the toxicity in mam
`mals, such as rats, pigs and dogs, and in primates, e.g.,
`baboons, was too severe to proceed to clinical phase
`trials (Collier, D. St. J., et al., Transplant. Proc, Vol.
`XX, No. 1, pp. 226-228, 1988).
`It would be extremely useful to discover a compound
`having immunosuppressive activity which could be
`employed to increase transplant acceptance in a recipi—
`ent but without causing serious toxic side effects typi
`cally associated with conventional immunosuppressant
`therapy, such as those discussed above.
`Rapamycin is a lipophilic macrolide with certain
`structural similarities to FK506 produced by Streptomy
`ces hygroscopicus with both antifungal and antitumor
`properties (Sehgal, S. N. et al., J. Antibiot., Vol. 28, pp.
`727-732, 1975; Eng. C. P., et al., J. Antibiar., Vol. 37, pp.
`1231-1237, 1984).
`It was reported that rapamycin inhibited two experi
`mental immunopathies, i.e., experimental allergic en
`cephalitis and adjuvant arthritis, and the formation of
`60
`humoral (IgE~like) antibody. (Martel, R. R., et al., Can.
`J. Physio. PharmacoL, 55: 48-51, 1977) It has also been
`reported recently that rapamycin inhibits murine T cell
`activation, apparently through a different mechanism
`from FK506 (Staruch, M. J., et al., The FASEB Journal,
`Vol 3, No. 3, abstract #3411, 1989). In addition, it was
`disclosed that rapamycin blocks the immunosuppressive
`effect of FK506 but not that of cyclosporin A (Dumont,
`
`45
`
`50
`
`55
`
`65
`
`2
`F. J. et al., The FASEB Journal, Vol. 3, No. 4, abstract
`#5256, 1989). There was no teaching or suggestion in
`these reports, however, that rapamycin could or should
`be used to effectively inhibit organ or tissue transplant
`rejection in mammals. Furthermore, these reports do
`not disclose or intimate that the toxic side-effects associ
`ated with FK506, and other immunosuppressive agents,
`would not likewise arise from administering rapamycin
`as an agent to inhibit transplant rejection in transplant
`operations.
`OBJECTS OF THE INVENTION
`It is an object of this invention to provide a method of
`increasing allograft acceptance (or inhibiting organ or
`tissue transplant rejection) in mammals by administer
`ing an efficacious compound of low toxicity.
`It is another object of this invention to reduce the
`toxicity of other conventional chemotherapeutic agents
`for inhibiting transplant rejection by combining their ~
`administration with an efficacious compound of low
`toxicity.
`These and other objects of the invention will become
`clearer in light of the detailed description which fol
`lows.
`
`SUMMARY OF THE INVENTION
`The present inventor has discovered a method of
`inhibiting organ or tissue transplant rejection in a mam
`mal in need thereof, comprising administering to said
`mammal a transplant rejection inhibiting amount of
`rapamycin.
`The present inventor has also discovered a method of
`inhibiting organ or tissue transplant rejection in a mam
`mal in need thereof, comprising administering to said
`mammal (a) an amount of rapamycin in combination
`with (b) an amount of one or more other chemothera
`peutic agents for inhibiting transplant rejection, said
`amounts of (a) and (b) being effective to inhibit trans
`plant rejection and to maintain inhibition of transplant
`rejection.
`DETAILED DESCRIPTION OF THE
`INVENTION
`All patents and literature references are hereby incor
`porated by reference in their entirety.
`The present invention provides a method of inhibit
`ing organ or tissue transplant rejection in a mammal in
`need thereof, comprising administering to said mammal
`a transplant rejection inhibiting amount of rapamycin.
`The present invention also provides a method of
`inhibiting organ or tissue transplant rejection in a mam
`mal in need thereof, comprising administering to said
`mammal an amount of rapamycin effective to inhibit
`transplant rejection and to maintain inhibition of trans
`plant rejection.
`As used herein, the terms “inhibiting organ or tissue
`transplant rejection” and “maintain inhibition of trans
`plant rejection" refer to increasing organ or tissue trans
`plant acceptance (or decreasing the likelihood of organ
`or tissue transplant rejection) involving allografts, i.e.,
`transplantation of organs or tissues from donor to recip
`ient both of whom are in the same species (intraspe
`cific), such as Homo sapiens.
`Rapamycin is an antifungal antibiotic which is ex
`tractable from a streptomycete, e. g., Streptomyces hygro
`scopicus. Methods for the preparation of rapamycin are
`disclosed in Sehga] et al., US. Pat. Nos. 3,929,992, and
`
`Ex. 1070-0002
`
`

`
`I5
`
`25
`
`35
`
`4-0
`
`20
`
`5,100,899
`3
`4
`3,993,749. In addition, monoacyl and diacyl derivatives
`ders for the preparation of sterile injectible solutions or
`of rapamycin and methods for their preparation are
`dispersions. In addition, rapamycin may be adminis
`disclosed by Rakhit, U.S. Pat. No. 4,316,885. Further
`tered in tablets, caplets, capsules, and the like for conve
`nient oral administration. Rapamycin may be adminis
`more, Stella et al., U.S. Pat. No. 4,650,803 disclose
`water soluble prodrugs of rapamycin, i.e., rapamycin
`tered in a pharmaceutically compatible or acceptable
`derivatives including the following rapamycin pro
`carrier, which includes by way of non-limiting example,
`drugs: glycinate prodrugs, propionate prodrugs and the
`oils, e.g., olive oil, alcohol, propylene glycol, and sur
`pyrrolidino butyrate prodrugs.
`factants, such as Chemophor EL (BASS).
`The methods and compositions of the present inven
`Another useful feature of this invention resides in the
`administration of rapamycin in combination with other
`tion include the use of natural and synthetic rapamycin,
`genetically engineered rapamycin and all derivatives
`conventional drug therapies, such as “triple therapy,“
`azathioprine (available from Burroughs Wellcome Co.,
`and prodrugs of rapamycin, such as described in the
`Research Triangle Park, N.C., under the tradename
`aforementioned U.S. patents, U.S. Pat. Nos. 3,929,992;
`Imuran ®), corticosteriods (available from the Upjohn
`3,993,749; 4,316,885; and 4,650,803, the contents of
`which are hereby incorporated by reference.
`Company, Kalamazoo, Michigan, under the tradename
`The present inventor has noted the efficacy of rapa
`Solu-Medrol ®); cyclosporin - (and cyclosporin A)
`mycin in inhibiting transplant rejection, e.g., by depress
`(available from Sandoz Pharmaceuticals, East Hanover,
`New Jersey, under the tradename Sandimmune ®), and
`ing the immune system in mammals without the atten
`also FK506, (available from Fujisawa Pharmaceutical
`dant toxic side-effects associated with other conven
`tional immunosuppressive agents, e.g., azathioprine,
`Co., Ltd., Osaka, Japan, under the tradename Fujimy
`corticosteroids and cyclosporin. Among such toxic
`cin ®). By combining rapamycin with such other con
`ventional chemotherapeutic drugs or agents for inhibit
`side-effects are nephrotoxicity, severe leukopenia,
`thrombocytopenia, Cushing‘s Syndrome and diabetes.
`ing transplant rejection, the toxicity of the latter may be
`advantageously reduced in that lesser amounts of such
`It has been discovered that rapamycin reduces or
`inhibits allograft rejection in mammals, i.e., organ or
`toxic drug or agents are required in order to inhibit
`transplant rejection in a mammalian transplant subject
`tissue transplantation from donor to recipient of the
`same species. Among such transplanted organs or tis
`either before or after transplantation, and also to main
`tain inhibition of transplant rejection.
`sues and given illustratively, are heart, liver, kidney,
`spleen, lung. small bowel, pancreas, and bone marrow,
`Thus, the present invention provides a method of
`inhibiting transplant rejection in a mammal in need
`or a combination of any of the foregoing.
`thereof, comprising administering to said mammal (a)
`As used herein, the term “transplant rejection inhibit~
`ing amount" refers to the amount of rapamycin (or of
`an amount of rapamycin in combination with (b) an
`rapamycin in combination with one or more other che
`amount of one or more other chemotherapeutic agents
`motherapeutic agents for inhibiting transplant rejection)
`for inhibiting transplant rejection, the amounts of (a)
`and (b) together being effective to inhibit transplant
`which may be administered so as to inhibit transplant
`rejection and to maintain inhibition of transplant rejec
`rejection in a mammal and to maintain transplant rejec
`tion inhibition, without causing severe toxic side-ef
`tion. It should be understood that the amount of either
`fects, e.g., nephrotoxicity, renal failure, etc. Those
`(a) or (b) alone, might or might not be effective in inhib
`iting and maintaining transplant rejection. The combi
`skilled in the art will appreciate that the dosage or
`amount of a transplant rejection inhibiting compound
`nation of the two components (a) and (b), together,
`however, is effective to inhibit transplant rejection and
`which is administered to a subject about to undergo or
`to maintain inhibition of transplant rejection.
`having undergone an organ or tissue transplant, will
`vary according to a number of factors, including indi
`Therefore, is used herein, such "other" chemothera
`peutic agents for inhibiting transplant rejection include,
`vidual characteristics, such as weight, age, and other
`for example, azathioprine, corticosteroids, cyclosporin
`factors, such as the type of organ or tissue transplanted
`or about to be transplanted.
`(and cyclosporin A), and FK506, or a combination of
`any of the foregoing.
`In one aspect of this invention, a tissue rejection in
`hibiting amount of rapamycin comprises from about 0.5
`The various features of the invention described
`to about 50 mg/kg/day, preferably from about l to
`above, such as the type of organ or tissue which is trans
`about 5 mg/kg/day. In another aspect, the inhibiting
`planted; the amount effective to inhibit transplant rejec
`transplant rejection amount of rapamycin is adminis
`tion and to maintain inhibition of transplant rejection;
`tered for a period of time comprising from about 1 to
`the mode or route of administration; and the duration of
`about 180 days, or longer, as necessary. Those skilled in
`treatment; apply to the method of inhibiting organ or
`tissue transplant rejection by administering rapamycin
`the art will recognize that compounds, drugs, agents,
`and the like, for inhibiting transplant rejection, may be
`in combination with one or more other chemotherapeu
`tic agents for inhibiting transplant rejection and to
`administered to a subject mammal, e.g., a human, for an
`maintain inhibition of transplant rejection.
`inde?nite post-transplantation period, in some instances,
`for the lifetime of the subject, provided, of course, that
`In addition, however, it should be understood that
`the subject is tolerating the compound, drug, agent,
`such other chemotherapeutic agents may be adminis
`tered continuously or intermittently with rapamycin.
`etc., reasonably well without serious side-effects.
`Rapamycin may be administered either orally or par
`Furthermore, the route of administration may differ
`enterally, e.g., intramuscularly, intraperitoneally, sub
`from that employed for rapamycin. That is to say, such
`other chemotherapeutic agents may be administered
`cutaneously, or intravenously to a mammal subject. The
`parenterally while rapamycin is being administered
`preferred route of administration is oral.
`orally to the mammalian subject.
`According to this invention, rapamycin may be ad
`ministered in various pharmaceutical forms, including
`The present invention is described below in speci?c
`pharmaceutical forms suitable for injectable use, such as
`working examples which are intended to illustrate the
`invention without limiting its scope.
`sterile aqueous solutions or dispersions and sterile pow
`
`45
`
`50
`
`65
`
`Ex. 1070-0003
`
`

`
`5
`EXAMPLE 1
`Organ Allograft Studies
`Rats: Heterotopic heart allografts were performed
`from DA donor to PVG recipients. DA and PVG refer
`to speci?c rat strains. Rapamycin was administered
`intramuscularly in olive oil for the ?rst l0 postoperative
`days except for Group 6 which only received the drug
`on days 3-6 postoperatively. Graft survival was as
`sessed by daily palpation.
`Large Animals: On the basis of the initial toxicity
`study two experiments were considered suitable to test
`whether rapamycin was immunosuppressivc, namely, a
`short term dosing in dogs following renal transplanta
`tion and inde?nite dosing of pigs, again following renal
`grafting.
`Pigs: In pigs an orthotopic renal transplantation
`model was used and Mixed Lymphocyte Cultures
`(MLC) were performed to con?rm histoincompatibility
`between donor and recipient. Untreated controls mean
`survival time is less than 10 days. Rapamycin was ad
`ministered at a dose of Zmg/kg orally daily commenc
`ing on the ?rst postoperative day.
`
`Results:
`
`Allograft Studies
`Rat:
`
`5,100,899
`6
`oil at a maximum concentration of 15 mg/ml and admin
`istered by daily intramuscular injection at dose sched
`ules varying from 0.5 mg/kg to 50 mg/kg for ten con
`secutive days and in the last group at 10 mg/kg on days
`3 to 6. Graft survival was assessed by daily palpation of
`the heart.
`Rapamycin prolonged allograft survival at all doses
`tested. Although there was some loss of weight this was
`not as marked as that found when FK506 was adminis
`tered to rats.
`Dogs
`In dogs dosing at all levels induced a vasculitis and at
`doses greater than 0.25 mg/kg this led to such severe
`manifestations that they were killed before the end of
`the 28 day study. At the higher doses the vasculitis
`affected the gastrointestinal tract, interestingly, it also
`caused a thrombocytopaenia. Marked depletion of cells
`in the lymphoid tissue, particularly B cells, occurred. In
`the dog, toxicity due to a vasculitis, that appears to have
`a particular predilection for the gastrointestinal tract,
`made it impossible to assess the immunosuppressive
`effects of the drug in this particular model. This species
`speci?c reaction to rapamycin con?rmed similar unpub
`lished observations of the inventor and his associates.
`Pigs
`Survival and cause of death are shown in Table 2
`with current creatinine values.
`TABLE 2
`SURVIVAL OF PIG ALLOGRAFI'S RECEIVING RAPAMYCIN 2 mg/Kg/DAY ORALLY IN DAYS AND
`CURRENT CREATININE
`
`20
`
`25
`
`SURVIVAL RENAL I-IISTOLOGY
`
`CAUSE OF
`CREATININE OUTCOME DEATH
`
`DAY LAST
`DOSED
`
`l
`2
`3
`4
`5
`6
`
`7
`
`8
`
`9
`
`10
`
`> 51
`> 66
`> 72
`4
`5
`48
`
`49
`
`5O
`
`55
`
`63
`
`ACUTE REJECTION
`INFARCTION
`FOCI PYELONEPHRITIS
`
`MILD ATN
`
`MILD ATN
`
`EARLY MILD REJECTION
`
`MODERATE ATN
`
`2 l 7
`I93
`283
`1,410
`
`I61
`
`235
`
`I76
`
`239
`
`2B3
`
`ALIVE
`ALIVE
`ALIVE
`DIED
`DIED
`DIED
`
`DIED
`
`DIED
`
`DIED
`
`DIED
`
`REJECTION
`INFARCTION
`INTERSTITIAL
`PNEUMONITIS
`INTERSTITIAL
`PNEUMONITIS
`INTERSTITIAL
`PNEUMONITIS
`INTERSTITIAL
`PNEUMONITIS
`INTERSTITIAL
`PNEUMONITIS
`
`43
`52
`64
`4
`5
`47
`
`48
`
`49
`
`$1
`
`62
`
`For the rat, survival of allografts is shown in Table 1.
`
`TABLE 1
`SURVIVAL OF HETEROTOPIC CARDIAC ALLOGRAFI‘S
`IN RATS
`Dose Schedule
`
`Group 1
`
`OM‘WN"
`
`No. Rats
`
`tlchbbbb
`
`Survival (days)
`75‘, 88', “XXX 2)
`50 mg/kg X 10 d
`65, 77. 88. I00
`10 mg/kg X 10 d
`2_ tug/kg X 10 d
`58. 59. 59. 66
`lmg/kg x 10d 34. 9. 52.
`4
`55
`35
`0.5 mg/kg X 10 d - I9. 20, 20,
`10 lug/kg d3-6
`I5. 19. 19,
`I9. 21
`NB. Rapamycin was administered intramuscularly in olive oil.
`(18 mg/ml and l0 mg/ml mpensions used.)
`NB. Rapamycin was administered intramuscularly in
`olive oil. (lSrng/ml and IOmg/ml suspensions used.)
`Rat strains used: DA donors in PVG recipients. con
`trol rejection time (n: l0)=7.4 days.
`Explanation of Table l: Heterotopic heart allografts
`in the neck of the rats were performed from DA donors
`to PVG recipients using the surgical techniques previ
`ously described (Heron, I., Acla, Pathol. Microbiol.
`Scand. 79:366, 1971). Rapamycin was dissolved in olive
`
`50
`
`55
`
`60
`
`65
`
`Explanation of Table 2: Orthotopic kidney transplan
`tation with contralateral nephrectomy was performed
`in the pig, as previously described (Calne, R. Y. et al.,
`Brit. J. Surg, 59: 969-977 (1972). Donor and recipient
`pairs were obtained from litters with different parents
`and incompatibility at the Major Histocampatibility
`Complex (MI-1C) was con?rmed by the mixed lympho
`cyte reaction (Bradley, B. A., et a1., Tissue Antigens, 4:
`283-290, 1974). Rapamycin was administered orally at 2
`mg/kg/day dissolved in olive oil at a concentration of
`10 mg/ml.
`In the case of pigs, one died of accelerated acute
`rejection and one died due to technical failure. The
`remaining eight animals recovered well and after an
`initial weight loss of approximately 10%. Subsequently,
`at about day 50 5 animals developed anorexia, diarrhea
`and became unwell to the extent that it was decided that
`they should be killed. Histological examination of these
`animals revealed that they were suffering from intersti
`tial pneumonitis, probably due to over-immunosuppres
`sion, and this was the reason that they became unwell.
`
`Ex. 1070-0004
`
`

`
`5,100,899
`7
`Furthermore, the renal histology did not show evidence
`of rejection except a mild degree in one animal who had
`not received the drug for 4 days. Histological examina
`tion of the colons in these animals showed mucosa] and
`submucosal edema but no vasculitis and no ulceration.
`Thus this was probably secondary to the systemic ef
`fects of the pneumonitis. The remaining three animals
`continued to thrive, all dosing having being stopped as
`indicated in Table 3.
`
`8
`In conclusion, rapamycin is a very effective immuno
`suppressive agent which can be employed to inhibit
`allograft transplantation rejection in mammalian sub
`jects.
`What is claimed is:
`1. A method of inhibiting organ or tissue transplant
`rejection in a mammal in need thereof, comprising ad
`ministrating to said mammal a transplant rejection in
`hibiting amount of rapamycin.
`2. The method according to claim 1, wherein a trans
`plant rejection inhibiting amount of rapamycin is ad
`ministered in an amount comprising from about 0.5 to
`about 50 mg/kg/day.
`3. The method according to claim 2, wherein said
`amount comprises from about 1 to about 5 mg/kg/day.
`4. The method according to claim 1, wherein said
`rapamycin is administered for a period of time compris
`ing from about 1 to about 180 days.
`5. The method according to claim 1, wherein said
`rapamycin is administered orally.
`6. The method according to claim 1, wherein said
`rapamycin is administered parenterally.
`7. The method according to claim 1, wherein said
`rapamycin is administered for an inde?nite period of
`time to maintain inhibition of transparent rejection.
`‘ t
`t
`' t
`
`Discussion
`Rapamycin was immunosuppressive and not toxic in
`the rat down to a dose of 0.5mg/ltg although the com
`pound was more effective at higher‘doses.
`In pigs, the results of the toxicity study showed that
`the drug was tolerated at a dose of 1 mg/kg in that both
`animals gained weight. On histological examination
`colitis was seen but no vasculitis was found or sug
`gested. Rapamycin was effective as an immunosuppres
`sive agent but after about 50 days of continuous closing
`at 2 mg/kg, 50% of the animals developed interstitial
`pneumonitis due to over-immunosuppression, and these
`animals were killed. However, none of the animals
`showed any evidence of ulceration in the colon or vas
`culitis. Therefore, in future studies monitoring of blood
`drug levels will be of bene?t.
`
`15
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`Ex. 1070-0005
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`
`PATENT NO. 1
`
`5,100,899
`
`DATED
`INVENTOR(S) I
`
`March 31, 1992
`Sir R. Calne
`
`It is certi?ed that error appears in the above-indenti?ed patent and that said Letters Patent is hereby
`corrected as shown below:
`
`Column 8, line 25:
`Claim 7,
`Change: "transparent" to ——transplant--.
`
`Signed and Sealed this
`
`Sixteenth Day of August, 1994
`
`Arresting O?icer
`
`Commissioner of Patents and Trademarks
`
`BRUCE LEHMAN
`
`Ex. 1070-0006